Methods for detecting modulators of cytokine receptor zalpha11

ABSTRACT

Novel polypeptides, polynucleotides encoding the polypeptides, and related compositions and methods are disclosed for zalpha11, a novel cytokine receptor. The polypeptides may be used within methods for detecting ligands that stimulate the proliferation and/or development of hematopoietic, lymphoid and myeloid cells in vitro and in vivo. Ligand-binding receptor polypeptides can also be used to block ligand activity in vitro and in vivo. The polynucleotides encoding zalpha11, are located on chromosome 16, and can be used to identify a region of the genome associated with human disease states. The present invention also includes methods for producing the protein, uses therefor and antibodies thereto.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/623,651, filed Nov. 23, 2009 now U.S. Pat. No. 7,923,212, which is acontinuation of U.S. patent application Ser. No. 11/537,835, filed Oct.2, 2006, issued as U.S. Pat. No. 7,638,286, which is a divisional ofU.S. patent application Ser. No. 10/715,998, filed Nov. 18, 2003, issuedas U.S. Pat. No. 7,411,056, which is a divisional of U.S. patentapplication Ser. No. 09/404,641, filed Sep. 23, 1999, issued as U.S.Pat. No. 6,576,744, which claims the benefit of U.S. Provisional PatentApplication No. 60/100,896, filed Sep. 23, 1998, U.S. Provisional PatentApplication 60/123,546, filed Mar. 9, 1999, and U.S. Provisional PatentApplication No. 60/142,574, filed Jul. 6, 1999, all of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

Proliferation and differentiation of cells of multicellular organismsare controlled by hormones and polypeptide growth factors. Thesediffusable molecules allow cells to communicate with each other and actin concert to form cells and organs, and to repair damaged tissue.Examples of hormones and growth factors include the steroid hormones(e.g. estrogen, testosterone), parathyroid hormone, follicle stimulatinghormone, the interleukins, platelet derived growth factor (PDGF),epidermal growth factor (EGF), granulocyte-macrophage colony stimulatingfactor (GM-CSF), erythropoietin (EPO) and calcitonin.

Hormones and growth factors influence cellular metabolism by binding toreceptors. Receptors may be integral membrane proteins that are linkedto signaling pathways within the cell, such as second messenger systems.Other classes of receptors are soluble molecules, such as thetranscription factors. Of particular interest are receptors forcytokines, molecules that promote the proliferation and/ordifferentiation of cells. Examples of cytokines include erythropoietin(EPO), which stimulates the development of red blood cells;thrombopoietin (TPO), which stimulates development of cells of themegakaryocyte lineage; and granulocyte-colony stimulating factor(G-CSF), which stimulates development of neutrophils. These cytokinesare useful in restoring normal blood cell levels in patients sufferingfrom anemia, thrombocytopenia, and neutropenia or receiving chemotherapyfor cancer.

The demonstrated in vivo activities of these cytokines illustrate theenormous clinical potential of, and need for, other cytokines, cytokineagonists, and cytokine antagonists. The present invention addressesthese needs by providing new a hematopoietic cytokine receptor, as wellas related compositions and methods.

The present invention provides such polypeptides for these and otheruses that should be apparent to those skilled in the art from theteachings herein.

SUMMARY OF THE INVENTION

Within one aspect, the present invention provides an isolatedpolynucleotide that encodes a zalpha11 polypeptide comprising a sequenceof amino acid residues that is at least 90% identical to an amino acidsequence selected from the group consisting of: (a) the amino acidsequence as shown in SEQ ID NO:2 from amino acid number 20 (Cys), toamino acid number 237 (His); (b) the amino acid sequence as shown in SEQID NO:2 from amino acid number 20 (Cys), to amino acid number 255 (Leu);(c) the amino acid sequence as shown in SEQ ID NO:2 from amino acidnumber 256 (Lys), to amino acid number 538 (Ser); (d) the amino acidsequence as shown in SEQ ID NO:2 from amino acid number 20 (Cys), toamino acid number 538 (Ser); and (e) the amino acid sequence as shown inSEQ ID NO:2 from amino acid number 1 (Met) to amino acid number 538(Ser), wherein the amino acid percent identity is determined using aFASTA program with ktup=1, gap opening penalty=10, gap extensionpenalty=1, and substitution matrix=BLOSUM62, with other parameters setas default. Within one embodiment, the isolated polynucleotide disclosedabove comprises a sequence of polynucleotides that is selected from thegroup consisting of: (a) a polynucleotide sequence as shown in SEQ IDNO:4 from nucleotide 1 to nucleotide 1614; (b) a polynucleotide sequenceas shown in SEQ ID NO:1 from nucleotide 126 to nucleotide 779; (c) apolynucleotide sequence as shown in SEQ ID NO:1 from nucleotide 126 tonucleotide 833; (d) a polynucleotide sequence as shown in SEQ ID NO:1from nucleotide 834 to nucleotide 1682; (e) a polynucleotide sequence asshown in SEQ ID NO:1 from nucleotide 126 to nucleotide 1682; and (f) apolynucleotide sequence as shown in SEQ ID NO:1 from nucleotide 69 tonucleotide 1682. Within another embodiment, the isolated polynucleotidedisclosed above comprises a sequence of amino acid residues selectedfrom the group consisting of: (a) the amino acid sequence as shown inSEQ ID NO:2 from amino acid number 20 (Cys), to amino acid number 237(His); (b) the amino acid sequence as shown in SEQ ID NO:2 from aminoacid number 20 (Cys), to amino acid number 255 (Leu); (c) the amino acidsequence as shown in SEQ ID NO:2 from amino acid number 256 (Lys), toamino acid number 538 (Ser); (d) the amino acid sequence as shown in SEQID NO:2 from amino acid number 20 (Cys), to amino acid number 538 (Ser);and (e) the amino acid sequence as shown in SEQ ID NO:2 from amino acidnumber 1 (Met) to amino acid number 538 (Ser). Within anotherembodiment, the isolated polynucleotide disclosed above consists of asequence of amino acid residues selected from the group consisting of:(a) the amino acid sequence as shown in SEQ ID NO:2 from amino acidnumber 20 (Cys), to amino acid number 237 (His); (b) the amino acidsequence as shown in SEQ ID NO:2 from amino acid number 20 (Cys), toamino acid number 255 (Leu); (c) the amino acid sequence as shown in SEQID NO:2 from amino acid number 256 (Lys), to amino acid number 538(Ser); (d) the amino acid sequence as shown in SEQ ID NO:2 from aminoacid number 20 (Cys), to amino acid number 538 (Ser); and (e) the aminoacid sequence as shown in SEQ ID NO:2 from amino acid number 1 (Met) toamino acid number 538 (Ser). Within another embodiment, the isolatedpolynucleotide disclosed above further comprises a WSWSX domain. Withinanother embodiment, the isolated polynucleotide disclosed above furthercomprises a transmembrane domain. Within another embodiment, theisolated polynucleotide disclosed above comprises a transmembrane domainconsisting of residues 238 (Leu) to 255 (Leu) of SEQ ID NO:2. Withinanother embodiment, the isolated polynucleotide disclosed above furthercomprises an intracellular domain. Within another embodiment, theisolated polynucleotide disclosed above comprises an intracellulardomain consists of residues 256 (Lys) to 538 (Ser) of SEQ ID NO:2.Within another embodiment, the isolated polynucleotide disclosed abovecomprises an intracellular domain which domain further comprises Box Iand Box II sites. comprises an intracellular domain wherein thepolypeptide further comprises an affinity tag.

Within a second aspect, the present invention provides an expressionvector comprising the following operably linked elements: atranscription promoter; a DNA segment encoding a zalpha11 polypeptidehaving an amino acid sequence as shown in SEQ ID NO:2 from amino acidnumber 20 (Cys), to amino acid number 538 (Ser); and a transcriptionterminator, wherein the promoter is operably linked to the DNA segment,and the DNA segment is operably linked to the transcription terminator.Within one embodiment, the expression vector disclosed above furthercomprises a secretory signal sequence operably linked to the DNAsegment.

Within a third aspect, the present invention provides a cultured cellcomprising an expression vector as disclosed above, wherein the cellexpresses a polypeptide encoded by the DNA segment.

Within a fourth aspect, the present invention provides an expressionvector comprising: a transcription promoter; a DNA segment encoding azalpha11 polypeptide having an amino acid sequence as shown in SEQ IDNO:2 from amino acid number 20 (Cys), to amino acid number 237 (His);and a transcription terminator, wherein the promoter, DNA segment, andterminator are operably linked. Within one embodiment, the expressionvector disclosed above further comprises a secretory signal sequenceoperably linked to the DNA segment. Within another embodiment, theexpression vector disclosed above further comprises a transmembranedomain operably linked to the DNA segment. Within another embodiment,the expression vector disclosed above further comprises a transmembranedomain consisting of residues 238(Leu) to 255 (Leu) of SEQ ID NO:2.Within another embodiment, the expression vector disclosed above furthercomprises an intracellular domain operably linked to the DNA segment.Within another embodiment, the expression vector disclosed above furthercomprises an intracellular domain consisting of residues 256 (Lys) to538 (Ser) of SEQ ID NO:2.

Within another aspect, the present invention provides a cultured cellinto which has been introduced an expression vector according to claim15, wherein the cell expresses a soluble receptor polypeptide encoded bythe DNA segment. Within one embodiment, the cultured cell disclosedabove is dependent upon an exogenously supplied hematopoietic growthfactor for proliferation.

Within another aspect, the present invention provides a DNA constructencoding a fusion protein, the DNA construct comprising: a first DNAsegment encoding a polypeptide having a sequence of amino acid residuesselected from the group consisting of: (a) the amino acid sequence ofSEQ ID NO:2 from amino acid number 1 (Met), to amino acid number 19(Gly); (b) the amino acid sequence of SEQ ID NO:2 from amino acid number20 (Cys) to amino acid number 237 (His); (c) the amino acid sequence ofSEQ ID NO:2 from amino acid number 20 (Cys) to amino acid number 255(Leu); (d) the amino acid sequence of SEQ ID NO:2 from amino acid number238 (Leu) to amino acid number 255 (Leu); (e) the amino acid sequence ofSEQ ID NO:2 from amino acid number 238 (Leu) to amino acid number 538(Ser); (f) the amino acid sequence of SEQ ID NO:2 from amino acid number256 (Lys) to amino acid number 538 (Ser); and (g) the amino acidsequence of SEQ ID NO:2 from amino acid number 20 (Cys), to amino acidnumber 538 (Ser); and at least one other DNA segment encoding anadditional polypeptide, wherein the first and other DNA segments areconnected in-frame; and wherein the first and other DNA segments encodethe fusion protein.

Within another aspect, the present invention provides an expressionvector comprising the following operably linked elements: atranscription promoter; a DNA construct encoding a fusion protein asdisclosed above; and a transcription terminator, wherein the promoter isoperably linked to the DNA construct, and the DNA construct is operablylinked to the transcription terminator.

Within another aspect, the present invention provides a cultured cellcomprising an expression vector as disclosed above, wherein the cellexpresses a polypeptide encoded by the DNA construct.

Within another aspect, the present invention provides a method ofproducing a fusion protein comprising: culturing a cell as disclosedabove; and isolating the polypeptide produced by the cell.

Within another aspect, the present invention provides an isolatedpolypeptide comprising a sequence of amino acid residues that is atleast 90% identical to an amino acid sequence selected from the groupconsisting of: (a) the amino acid sequence as shown in SEQ ID NO:2 fromamino acid number 20 (Cys), to amino acid number 237 (His); (b) theamino acid sequence as shown in SEQ ID NO:2 from amino acid number 20(Cys), to amino acid number 255 (Leu); (c) the amino acid sequence asshown in SEQ ID NO:2 from amino acid number 256 (Lys), to amino acidnumber 538 (Ser); (d) the amino acid sequence as shown in SEQ ID NO:2from amino acid number 20 (Cys), to amino acid number 538 (Ser); and (e)the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 1(Met) to amino acid number 538 (Ser), wherein the amino acid percentidentity is determined using a FASTA program with ktup=1, gap openingpenalty=10, gap extension penalty=1, and substitution matrix=BLOSUM62,with other parameters set as default. Within one embodiment, theisolated polypeptide disclosed above comprises a sequence of amino acidresidues selected from the group consisting of: (a) the amino acidsequence as shown in SEQ ID NO:2 from amino acid number 20 (Cys), toamino acid number 237 (His); (b) the amino acid sequence as shown in SEQID NO:2 from amino acid number 20 (Cys), to amino acid number 255 (Leu);(c) the amino acid sequence as shown in SEQ ID NO:2 from amino acidnumber 256 (Lys), to amino acid number 538 (Ser); (d) the amino acidsequence as shown in SEQ ID NO:2 from amino acid number 20 (Cys), toamino acid number 538 (Ser); and (e) the amino acid sequence as shown inSEQ ID NO:2 from amino acid number 1 (Met) to amino acid number 538(Ser). Within another embodiment, the isolated polypeptide disclosedabove consists of a sequence of amino acid residues selected from thegroup consisting of: (a) the amino acid sequence as shown in SEQ ID NO:2from amino acid number 20 (Cys), to amino acid number 237 (His); (b) theamino acid sequence as shown in SEQ ID NO:2 from amino acid number 20(Cys), to amino acid number 255 (Leu); (c) the amino acid sequence asshown in SEQ ID NO:2 from amino acid number 256 (Lys), to amino acidnumber 538 (Ser); (d) the amino acid sequence as shown in SEQ ID NO:2from amino acid number 20 (Cys), to amino acid number 538 (Ser); and (e)the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 1(Met) to amino acid number 538 (Ser). Within another embodiment, theisolated polypeptide disclosed above further contains a WSXWS motif.Within another embodiment, the isolated polypeptide disclosed abovefurther comprises a transmembrane domain. Within another embodiment, theisolated polypeptide disclosed above further comprises a transmembranedomain, wherein the transmembrane domain consists of residues 238(Leu)to 255 (Leu) of SEQ ID NO:2. Within another embodiment, the isolatedpolypeptide disclosed above further comprises an intracellular domain.Within another embodiment, the isolated polypeptide disclosed abovefurther comprises an intracellular domain, wherein the intracellulardomain consists of residues 256 (Lys) to 538 (Ser) of SEQ ID NO:2.Within another embodiment, the isolated polypeptide disclosed abovefurther comprises an intracellular domain, wherein the intracellulardomain further comprises Box I and Box II sites.

Within another aspect, the present invention provides a method ofproducing a zalpha11 polypeptide comprising: culturing a cell asdisclosed above; and isolating the zalpha11 polypeptide produced by thecell.

Within another aspect, the present invention provides an isolatedpolypeptide comprising an amino acid sequence selected from the groupconsisting of: (a) the amino acid sequence as shown in SEQ ID NO:2 fromamino acid number 20 (Cys), to amino acid number 237 (His); and whereinthe polypeptide is substantially free of transmembrane and intracellulardomains ordinarily associated with hematopoietic receptors. Withinanother embodiment, the isolated polypeptide disclosed above comprisesan affinity tag.

Within another aspect, the present invention provides a method ofproducing a zalpha11 polypeptide comprising: culturing a cell asdisclosed above; and isolating the zalpha11 polypeptide produced by thecell.

Within another aspect, the present invention provides a method ofproducing an antibody to zalpha11 polypeptide comprising: inoculating ananimal with a polypeptide selected from the group consisting of: (a) apolypeptide consisting of 9 to 519 amino acids, wherein the polypeptideconsists of a contiguous sequence of amino acids in SEQ ID NO:2 fromamino acid number 20 (Cys), to amino acid number 538 (Ser); (b) apolypeptide consisting of the amino acid sequence of SEQ ID NO:2 fromamino acid number 20 (Cys), to amino acid number 237 (His); (c) apolypeptide consisting of the amino acid sequence of SEQ ID NO:2 fromamino acid number 101 (Leu) to amino acid number 122 (Gly); (d) apolypeptide consisting of the amino acid sequence of SEQ ID NO:2 fromamino acid number 141 (Asn) to amino acid number 174 (Ala); (e) apolypeptide consisting of the amino acid sequence of SEQ ID NO:2 fromamino acid number 193 (Cys) to amino acid number 261 (Val); (f) apolypeptide consisting of the amino acid sequence of SEQ ID NO:2 fromamino acid number 51 (Trp) to amino acid number 61 (Glu); (g) apolypeptide consisting of the amino acid sequence of SEQ ID NO:2 fromamino acid 136 (Ile) to amino acid number 143 (Glu); (h) a polypeptideconsisting of the amino acid sequence of SEQ ID NO:2 from amino acid 187(Pro) to amino acid number 195 (Ser); (i) a polypeptide consisting ofthe amino acid sequence of SEQ ID NO:2 from amino acid number 223 (Phe)to amino acid number 232 (Glu); and (j) a polypeptide consisting of theamino acid sequence of SEQ ID NO:2 from amino acid number 360 (Glu) toamino acid number 368 (Asp); and wherein the polypeptide elicits animmune response in the animal to produce the antibody; and isolating theantibody from the animal.

Within another aspect, the present invention provides an antibodyproduced by the method disclosed above, which specifically binds to azalpha11 polypeptide. Within one embodiment, the antibody disclosedabove is a monoclonal antibody.

Within another aspect, the present invention provides an antibody whichspecifically binds to a polypeptide as disclosed above.

Within another aspect, the present invention provides a method ofdetecting, in a test sample, the presence of a modulator of zalpha11protein activity, comprising: culturing a cell into which has beenintroduced an expression vector as disclosed above, wherein the cellexpresses the zalpha11 protein encoded by the DNA segment in thepresence and absence of a test sample; and comparing levels of activityof zalpha11 in the presence and absence of a test sample, by abiological or biochemical assay; and determining from the comparison,the presence of modulator of zalpha11 activity in the test sample.

Within another aspect, the present invention provides a method fordetecting a zalpha11 receptor ligand within a test sample, comprising:contacting a test sample with a polypeptide comprising an amino acidsequence as shown in SEQ ID NO:2 from amino acid number 20 (Cys), toamino acid number 237 (His); and detecting the binding of thepolypeptide to a ligand in the sample. Within one embodiment, the methoddisclosed above further comprises a polypeptide comprising transmembraneand intracellular domains. Within another embodiment, the methoddisclosed above further comprises a polypeptide wherein the polypeptideis membrane bound within a cultured cell, and the detecting stepcomprises measuring a biological response in the cultured cell. Withinanother embodiment, the method disclosed above further comprises apolypeptide wherein the polypeptide is membrane bound within a culturedcell, and the detecting step comprises measuring a biological responsein the cultured cell, wherein the biological response is cellproliferation or activation of transcription of a reporter gene. Withinanother embodiment, the method disclosed above further comprises apolypeptide wherein the polypeptide is immobilized on a solid support.

These and other aspects of the invention will become evident uponreference to the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a Hopp/Woods hydrophilicity plot of human zalpha11.

FIG. 2 is an alignment of human zalpha11 (zalpha) (SEQ ID NO: 2) andmouse zalpha11 (muzalp) (SEQ ID NO: 85).

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention in detail, it may be helpful to theunderstanding thereof to define the following terms:

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985),substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-10, 1988),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2: 95-107, 1991. DNAs encoding affinity tags are availablefrom commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M⁻¹.

The term “complements of a polynucleotide molecule” is a polynucleotidemolecule having a complementary base sequence and reverse orientation ascompared to a reference sequence. For example, the sequence 5′ ATGCACGGG3′ is complementary to 5′CCCGTGCAT 3′.

The term “contig” denotes a polynucleotide that has a contiguous stretchof identical or complementary sequence to another polynucleotide.Contiguous sequences are said to “overlap” a given stretch ofpolynucleotide sequence either in their entirety or along a partialstretch of the polynucleotide. For example, representative contigs tothe polynucleotide sequence 5′-ATGGCTTAGCTT-3′ are 5′-TAGCTTgagtct-3′and 3′-gtcgacTACCGA-5′.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

The term “expression vector” is used to denote a DNA molecule, linear orcircular, that comprises a segment encoding a polypeptide of interestoperably linked to additional segments that provide for itstranscription. Such additional segments include promoter and terminatorsequences, and may also include one or more origins of replication, oneor more selectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

The term “isolated”, when applied to a polynucleotide, denotes that thepolynucleotide has been removed from its natural genetic milieu and isthus free of other extraneous or unwanted coding sequences, and is in aform suitable for use within genetically engineered protein productionsystems. Such isolated molecules are those that are separated from theirnatural environment and include cDNA and genomic clones. Isolated DNAmolecules of the present invention are free of other genes with whichthey are ordinarily associated, but may include naturally occurring 5′and 3′ untranslated regions such as promoters and terminators. Theidentification of associated regions will be evident to one of ordinaryskill in the art (see for example, Dynan and Tijan, Nature 316:774-78,1985).

An “isolated” polypeptide or protein is a polypeptide or protein that isfound in a condition other than its native environment, such as apartfrom blood and animal tissue. In a preferred form, the isolatedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin. It is preferred to provide thepolypeptides in a highly purified form, i.e. greater than 95% pure, morepreferably greater than 99% pure. When used in this context, the term“isolated” does not exclude the presence of the same polypeptide inalternative physical forms, such as dimers or alternatively glycosylatedor derivatized forms.

The term “operably linked”, when referring to DNA segments, indicatesthat the segments are arranged so that they function in concert fortheir intended purposes, e.g., transcription initiates in the promoterand proceeds through the coding segment to the terminator.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

“Paralogs” are distinct but structurally related proteins made by anorganism. Paralogs are believed to arise through gene duplication. Forexample, α-globin, β-globin, and myoglobin are paralogs of each other.

A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides”.

The term “promoter” is used herein for its art-recognized meaning todenote a portion of a gene containing DNA sequences that provide for thebinding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes.

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

The term “receptor” is used herein to denote a cell-associated protein,or a polypeptide subunit of such a protein, that binds to a bioactivemolecule (the “ligand”) and mediates the effect of the ligand on thecell. Binding of ligand to receptor results in a conformational changein the receptor (and, in some cases, receptor multimerization, i.e.,association of identical or different receptor subunits) that causesinteractions between the effector domain(s) and other molecule(s) in thecell. These interactions in turn lead to alterations in the metabolismof the cell. Metabolic events that are linked to receptor-ligandinteractions include gene transcription, phosphorylation,dephosphorylation, cell proliferation, increases in cyclic AMPproduction, mobilization of cellular calcium, mobilization of membranelipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis ofphospholipids. Cell-surface cytokine receptors are characterized by amulti-domain structure as discussed in more detail below. Thesereceptors are anchored in the cell membrane by a transmembrane domaincharacterized by a sequence of hydrophobic amino acid residues(typically about 21-25 residues), which is commonly flanked bypositively charged residues (Lys or Arg). In general, receptors can bemembrane bound, cytosolic or nuclear; monomeric (e.g., thyroidstimulating hormone receptor, beta-adrenergic receptor) or multimeric(e.g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSFreceptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor).The term “receptor polypeptide” is used to denote complete receptorpolypeptide chains and portions thereof, including isolated functionaldomains (e.g., ligand-binding domains).

A “secretory signal sequence” is a DNA sequence that encodes apolypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger peptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

A “soluble receptor” is a receptor polypeptide that is not bound to acell membrane. Soluble receptors are most commonly ligand-bindingreceptor polypeptides that lack transmembrane and cytoplasmic domains.Soluble receptors can comprise additional amino acid residues, such asaffinity tags that provide for purification of the polypeptide orprovide sites for attachment of the polypeptide to a substrate, orimmunoglobulin constant region sequences. Many cell-surface receptorshave naturally occurring, soluble counterparts that are produced byproteolysis. Soluble receptor polypeptides are said to be substantiallyfree of transmembrane and intracellular polypeptide segments when theylack sufficient portions of these segments to provide membrane anchoringor signal transduction, respectively.

The term “splice variant” is used herein to denote alternative forms ofRNA transcribed from a gene. Splice variation arises naturally throughuse of alternative splicing sites within a transcribed RNA molecule, orless commonly between separately transcribed RNA molecules, and mayresult in several mRNAs transcribed from the same gene. Splice variantsmay encode polypeptides having altered amino acid sequence. The termsplice variant is also used herein to denote a protein encoded by asplice variant of an mRNA transcribed from a gene.

Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

All references cited herein are incorporated by reference in theirentirety.

The present invention is based in part upon the discovery of a novel DNAsequence that encodes a protein having the structure of a class Icytokine receptor. The deduced amino acid sequence indicated that theencoded receptor belongs to the receptor subfamily that includes theIL-2 receptor β-subunit and the β-common receptor (i.e., IL3, IL-5, andGM-CSF receptor β-subunits). Analysis of the tissue distribution of themRNA corresponding to this novel DNA showed expression in lymph node,peripheral blood leukocytes (PBLs), spleen, and thymus. Moreover, themRNA was abundant in the Raji cell line (ATCC No. CCL-86) derived from aBurkitt's lymphoma. The polypeptide has been designated zalpha11.

The novel zalpha11 polypeptides of the present invention were initiallyidentified by querying an EST database. An EST was found and itscorresponding cDNA was sequenced. The novel polypeptide encoded by thecDNA showed homology with class I cytokine receptors. The zalpha11polynucleotide sequence encodes the entire coding sequence of thepredicted protein. Zalpha11 is a novel cytokine receptor that may beinvolved in an apoptotic cellular pathway, cell-cell signaling molecule,growth factor receptor, or extracellular matrix associated protein withgrowth factor hormone activity, or the like.

The sequence of the zalpha11 polypeptide was deduced from a single clonethat contained its corresponding polynucleotide sequence. The clone wasobtained from a spinal cord library. Other libraries that might also besearched for such sequences include PBL, thymus, spleen, lymph node,human erythroleukemia cell lines (e.g., TF-1), Raji cells, acutemonocytic leukemia cell lines, other lymphoid and hematopoietic celllines, and the like.

The nucleotide sequence of a representative zalpha11-encoding DNA isdescribed in SEQ ID NO:1 (from nucleotide 69 to 1682), and its deduced538 amino acid sequence is described in SEQ ID NO:2. In its entirety,the zalpha11 polypeptide (SEQ ID NO:2) represents a full-lengthpolypeptide segment (residue 1 (Met) to residue 538 (Ser) of SEQ IDNO:2). The domains and structural features of the zalpha11 polypeptideare further described below.

Analysis of the zalpha11 polypeptide encoded by the DNA sequence of SEQID NO:1 revealed an open reading frame encoding 538 amino acids (SEQ IDNO:2) comprising a predicted secretory signal peptide of 19 amino acidresidues (residue 1 (Met) to residue 19 (Gly) of SEQ ID NO:2), and amature polypeptide of 519 amino acids (residue 20 (Cys) to residue 538(Ser) of SEQ ID NO:2). In addition to the WSXWS motif (SEQ ID NO:3)corresponding to residues 214 to 218 of SEQ ID NO:2, the receptorcomprises a cytokine-binding domain of approximately 200 amino acidresidues (residues 20 (Cys) to 237 (His) of SEQ ID NO:2); a domainlinker (residues 120 (Pro) to 123 (Pro) of SEQ ID NO:2); a penultimatestrand region (residues 192 (Lys) to 202 (Ala) of SEQ ID NO:2); atransmembrane domain (residues 238 (Leu) to 255 (Leu) of SEQ ID NO:2);complete intracellular signaling domain (residues 256 (Lys) to 538 (Ser)of SEQ ID NO:2) which contains a “Box I” signaling site (residues 267(Ile) to 273 (Pro) of SEQ ID NO:2), and a “Box II” signaling site(residues 301 (Leu) to 304 (Gly) of SEQ ID NO:2). Those skilled in theart will recognize that these domain boundaries are approximate, and arebased on alignments with known proteins and predictions of proteinfolding. In addition to these domains, conserved receptor features inthe encoded receptor include (as shown in SEQ ID NO:2) a conserved Trpresidue at position 138, and a conserved Arg residue at position 201.The corresponding polynucleotides encoding the zalpha11 polypeptideregions, domains, motifs, residues and sequences described above are asshown in SEQ ID NO:1.

The presence of transmembrane regions, and conserved and low variancemotifs generally correlates with or defines important structural regionsin proteins. Regions of low variance (e.g., hydrophobic clusters) aregenerally present in regions of structural importance (Sheppard, P. etal., supra.). Such regions of low variance often contain rare orinfrequent amino acids, such as Tryptophan. The regions flanking andbetween such conserved and low variance motifs may be more variable, butare often functionally significant because they may relate to or defineimportant structures and activities such as binding domains, biologicaland enzymatic activity, signal transduction, cell-cell interaction,tissue localization domains and the like.

The regions of conserved amino acid residues in zalpha11, describedabove, can be used as tools to identify new family members. Forinstance, reverse transcription-polymerase chain reaction (RT-PCR) canbe used to amplify sequences encoding the conserved regions from RNAobtained from a variety of tissue sources or cell lines. In particular,highly degenerate primers designed from the zalpha11 sequences areuseful for this purpose. Designing and using such degenerate primers maybe readily performed by one of skill in the art.

The present invention provides polynucleotide molecules, including DNAand RNA molecules, that encode the zalpha11 polypeptides disclosedherein. Those skilled in the art will recognize that, in view of thedegeneracy of the genetic code, considerable sequence variation ispossible among these polynucleotide molecules. SEQ ID NO:4 is adegenerate DNA sequence that encompasses all DNAs that encode thezalpha11 polypeptide of SEQ ID NO:2. Those skilled in the art willrecognize that the degenerate sequence of SEQ ID NO:4 also provides allRNA sequences encoding SEQ ID NO:2 by substituting U for T. Thus,zalpha11 polypeptide-encoding polynucleotides comprising nucleotide 1 tonucleotide 1614 of SEQ ID NO:4 and their RNA equivalents arecontemplated by the present invention. Table 1 sets forth the one-lettercodes used within SEQ ID NO:4 to denote degenerate nucleotide positions.“Resolutions” are the nucleotides denoted by a code letter. “Complement”indicates the code for the complementary nucleotide(s). For example, thecode Y denotes either C or T, and its complement R denotes A or G, Abeing complementary to T, and G being complementary to C.

TABLE 1 Nucleotide Resolution Complement Resolution A A T T C C G G G GC C T T A A R A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|GW A|T W A|T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T HA|C|T N A|C|G|T N A|C|G|T

The degenerate codons used in SEQ ID NO:4, encompassing all possiblecodons for a given amino acid, are set forth in Table 2.

TABLE 2 Amino One Letter  Degenerate Acid Code Codons Codon Cys CTGC TGT TGY Ser S AGC AGT TCA TCC WSN TCG TCT Thr T ACA ACC ACG ACT ACNPro P CCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly GGGA GGC GGG GGT GGN Asn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAGGAR Gln Q CAA CAG CAR His H CAC CAT CAY Arg R AGA AGG CGA CGC MGNCGG CGT Lys K AAA AAG AAR Met M ATG ATG Ile I ATA ATC ATT ATH Leu LCTA CTC CTG CTT YTN TTA TTG Val V GTA GTC GTG GTT GTN Phe F TTC TTT TTYTyr Y TAC TAT TAY Trp W TGG TGG Ter . TAA TAG TGA TRR Asn|Asp B RAYGlu|Gln Z SAR Any X NNN

One of ordinary skill in the art will appreciate that some ambiguity isintroduced in determining a degenerate codon, representative of allpossible codons encoding each amino acid. For example, the degeneratecodon for serine (WSN) can, in some circumstances, encode arginine(AGR), and the degenerate codon for arginine (MGN) can, in somecircumstances, encode serine (AGY). A similar relationship existsbetween codons encoding phenylalanine and leucine. Thus, somepolynucleotides encompassed by the degenerate sequence may encodevariant amino acid sequences, but one of ordinary skill in the art caneasily identify such variant sequences by reference to the amino acidsequence of SEQ ID NO:2. Variant sequences can be readily tested forfunctionality as described herein.

One of ordinary skill in the art will also appreciate that differentspecies can exhibit “preferential codon usage.” In general, see,Grantham, et al., Nuc. Acids Res. 8:1893-912, 1980; Haas, et al. Curr.Biol. 6:315-24, 1996; Wain-Hobson, et al., Gene 13:355-64, 1981;Grosjean and Fiers, Gene 18:199-209, 1982; Holm, Nuc. Acids Res.14:3075-87, 1986; Ikemura, J. Mol. Biol. 158:573-97, 1982. As usedherein, the term “preferential codon usage” or “preferential codons” isa term of art referring to protein translation codons that are mostfrequently used in cells of a certain species, thus favoring one or afew representatives of the possible codons encoding each amino acid (SeeTable 2). For example, the amino acid Threonine (Thr) may be encoded byACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonlyused codon; in other species, for example, insect cells, yeast, virusesor bacteria, different Thr codons may be preferential. Preferentialcodons for a particular species can be introduced into thepolynucleotides of the present invention by a variety of methods knownin the art. Introduction of preferential codon sequences intorecombinant DNA can, for example, enhance production of the protein bymaking protein translation more efficient within a particular cell typeor species. Therefore, the degenerate codon sequence disclosed in SEQ IDNO:4 serves as a template for optimizing expression of polynucleotidesin various cell types and species commonly used in the art and disclosedherein. Sequences containing preferential codons can be tested andoptimized for expression in various species, and tested forfunctionality as disclosed herein.

Within preferred embodiments of the invention the isolatedpolynucleotides will hybridize to similar sized regions of SEQ ID NO:1,or a sequence complementary thereto, under stringent conditions. Ingeneral, stringent conditions are selected to be about 5° C. lower thanthe thermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence hybridizes to aperfectly matched probe. Numerous equations for calculating T_(m) areknown in the art, and are specific for DNA, RNA and DNA-RNA hybrids andpolynucleotide probe sequences of varying length (see, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition(Cold Spring Harbor Press 1989); Ausubel et al., (eds.), CurrentProtocols in Molecular Biology (John Wiley and Sons, Inc. 1987); Bergerand Kimmel (eds.), Guide to Molecular Cloning Techniques, (AcademicPress, Inc. 1987); and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227(1990)). Sequence analysis software such as OLIGO 6.0 (LSR; Long Lake,Minn.) and Primer Premier 4.0 (Premier Biosoft International; Palo Alto,Calif.), as well as sites on the Internet, are available tools foranalyzing a given sequence and calculating T_(m) based on user definedcriteria. Such programs can also analyze a given sequence under definedconditions and identify suitable probe sequences. Typically,hybridization of longer polynucleotide sequences (e.g., >50 base pairs)is performed at temperatures of about 20-25° C. below the calculatedT_(m). For smaller probes (e.g., <50 base pairs) hybridization istypically carried out at the T_(m) or 5-10° C. below. This allows forthe maximum rate of hybridization for DNA-DNA and DNA-RNA hybrids.Higher degrees of stringency at lower temperatures can be achieved withthe addition of formamide which reduces the T_(m) of the hybrid about 1°C. for each 1% formamide in the buffer solution. Suitable stringenthybridization conditions are equivalent to about a 5 h to overnightincubation at about 42° C. in a solution comprising: about 40-50%formamide, up to about 6×SSC, about 5×Denhardt's solution, zero up toabout 10% dextran sulfate, and about 10-20 μg/ml denaturedcommercially-available carrier DNA. Generally, such stringent conditionsinclude temperatures of 20-70° C. and a hybridization buffer containingup to 6×SSC and 0-50% formamide; hybridization is then followed bywashing filters in up to about 2×SSC. For example, a suitable washstringency is equivalent to 0.1×SSC to 2×SSC, 0.1% SDS, at 55° C. to 65°C. Different degrees of stringency can be used during hybridization andwashing to achieve maximum specific binding to the target sequence.Typically, the washes following hybridization are performed atincreasing degrees of stringency to remove non-hybridized polynucleotideprobes from hybridized complexes. Stringent hybridization and washconditions depend on the length of the probe, reflected in the Tm,hybridization and wash solutions used, and are routinely determinedempirically by one of skill in the art.

As previously noted, the isolated polynucleotides of the presentinvention include DNA and RNA. Methods for preparing DNA and RNA arewell known in the art. In general, RNA is isolated from a tissue or cellthat produces large amounts of zalpha11 RNA. Such tissues and cells areidentified by Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA77:5201, 1980), and include PBLs, spleen, thymus, and lymph tissues,Raji cells, human erythroleukemia cell lines (e.g., TF-1), acutemonocytic leukemia cell lines, other lymphoid and hematopoietic celllines, and the like. Total RNA can be prepared using guanidiniumisothiocyanate extraction followed by isolation by centrifugation in aCsCl gradient (Chirgwin et al., Biochemistry 18:52-94, 1979). Poly (A)⁺RNA is prepared from total RNA using the method of Aviv and Leder (Proc.Natl. Acad. Sci. USA 69:1408-12, 1972). Complementary DNA (cDNA) isprepared from poly(A)⁺ RNA using known methods. In the alternative,genomic DNA can be isolated. Polynucleotides encoding zalpha11polypeptides are then identified and isolated by, for example,hybridization or polymerase chain reaction (PCR) (Mullis, U.S. Pat. No.4,683,202).

A full-length clone encoding zalpha11 can be obtained by conventionalcloning procedures. Complementary DNA (cDNA) clones are preferred,although for some applications (e.g., expression in transgenic animals)it may be preferable to use a genomic clone, or to modify a cDNA cloneto include at least one genomic intron. Methods for preparing cDNA andgenomic clones are well known and within the level of ordinary skill inthe art, and include the use of the sequence disclosed herein, or partsthereof, for probing or priming a library. Expression libraries can beprobed with antibodies to zalpha11, receptor fragments, or otherspecific binding partners.

The polynucleotides of the present invention can also be synthesizedusing DNA synthesis machines. Currently the method of choice is thephosphoramidite method. If chemically synthesized double stranded DNA isrequired for an application such as the synthesis of a gene or a genefragment, then each complementary strand is made separately. Theproduction of short polynucleotides (60 to 80 bp) is technicallystraightforward and can be accomplished by synthesizing thecomplementary strands and then annealing them. However, for producinglonger polynucleotides (>300 bp), special strategies are usuallyemployed, because the coupling efficiency of each cycle during chemicalDNA synthesis is seldom 100%. To overcome this problem, synthetic genes(double-stranded) are assembled in modular form from single-strandedfragments that are from 20 to 100 nucleotides in length.

One method for building a synthetic gene requires the initial productionof a set of overlapping, complementary oligonucleotides, each of whichis between 20 to 60 nucleotides long. Each internal section of the genehas complementary 3′ and 5′ terminal extensions designed to base pairprecisely with an adjacent section. Thus, after the gene is assembled,process is completed by sealing the nicks along the backbones of the twostrands with T4 DNA ligase. In addition to the protein coding sequence,synthetic genes can be designed with terminal sequences that facilitateinsertion into a restriction endonuclease site of a cloning vector.Moreover, other sequences should can be added that contain signals forproper initiation and termination of transcription and translation.

An alternative way to prepare a full-length gene is to synthesize aspecified set of overlapping oligonucleotides (40 to 100 nucleotides).After the 3′ and 5′ short overlapping complementary regions (6 to 10nucleotides) are annealed, large gaps still remain, but the shortbase-paired regions are both long enough and stable enough to hold thestructure together. The gaps are filled and the DNA duplex is completedvia enzymatic DNA synthesis by E. coli DNA polymerase I. After theenzymatic synthesis is completed, the nicks are sealed with T4 DNAligase. Double-stranded constructs are sequentially linked to oneanother to form the entire gene sequence which is verified by DNAsequence analysis. See Glick and Pasternak, Molecular Biotechnology,Principles & Applications of Recombinant DNA, (ASM Press, Washington,D.C. 1994); Itakura et al., Annu. Rev. Biochem. 53: 323-56, 1984 andClimie et al., Proc. Natl. Acad. Sci. USA 87:633-7, 1990.

The present invention further provides counterpart polypeptides andpolynucleotides from other species (orthologs). These species include,but are not limited to mammalian, avian, amphibian, reptile, fish,insect and other vertebrate and invertebrate species. Of particularinterest are zalpha11 polypeptides from other mammalian species,including murine, porcine, ovine, bovine, canine, feline, equine, andother primate polypeptides. Orthologs of human zalpha11 can be clonedusing information and compositions provided by the present invention incombination with conventional cloning techniques. For example, a cDNAcan be cloned using mRNA obtained from a tissue or cell type thatexpresses zalpha11 as disclosed herein. Suitable sources of mRNA can beidentified by probing Northern blots with probes designed from thesequences disclosed herein. A library is then prepared from mRNA of apositive tissue or cell line. A zalpha11-encoding cDNA can then beisolated by a variety of methods, such as by probing with a complete orpartial human cDNA or with one or more sets of degenerate probes basedon the disclosed sequences. A cDNA can also be cloned using PCR (Mullis,supra.), using primers designed from the representative human zalpha11sequence disclosed herein. Within an additional method, the cDNA librarycan be used to transform or transfect host cells, and expression of thecDNA of interest can be detected with an antibody to zalpha11polypeptide. Similar techniques can also be applied to the isolation ofgenomic clones.

Cytokine receptor subunits are characterized by a multi-domain structurecomprising an extracellular domain, a transmembrane domain that anchorsthe polypeptide in the cell membrane, and an intracellular domain. Theextracellular domain may be a ligand-binding domain, and theintracellular domain may be an effector domain involved in signaltransduction, although ligand-binding and effector functions may resideon separate subunits of a multimeric receptor. The ligand-binding domainmay itself be a multi-domain structure. Multimeric receptors includehomodimers (e.g., PDGF receptor αα and ββ isoforms, erythropoietinreceptor, MPL, and G-CSF receptor), heterodimers whose subunits eachhave ligand-binding and effector domains (e.g., PDGF receptor αβisoform), and multimers having component subunits with disparatefunctions (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, and GM-CSFreceptors). Some receptor subunits are common to a plurality ofreceptors. For example, the AIC2B subunit, which cannot bind ligand onits own but includes an intracellular signal transduction domain, is acomponent of IL-3 and GM-CSF receptors. Many cytokine receptors can beplaced into one of four related families on the basis of the structureand function. Hematopoietic receptors, for example, are characterized bythe presence of a domain containing conserved cysteine residues and theWSXWS motif (SEQ ID NO:3). Cytokine receptor structure has been reviewedby Urdal, Ann Reports Med. Chem. 26:221-228, 1991 and Cosman, Cytokine5:95-106, 1993. Under selective pressure for organisms to acquire newbiological functions, new receptor family members likely arise fromduplication of existing receptor genes leading to the existence ofmulti-gene families. Family members thus contain vestiges of theancestral gene, and these characteristic features can be exploited inthe isolation and identification of additional family members. Thus, thecytokine receptor superfamily is subdivided into several families, forexample, the immunoglobulin family (including CSF-1, MGF, IL-1, and PDGFreceptors); the hematopoietin family (including IL-2 receptor β-subunit,GM-CSF receptor α-subunit, GM-CSF receptor β-subunit; and G-CSF, EPO,IL-3, IL-4, IL-5, IL-6, IL-7, and IL-9 receptors); TNF receptor family(including TNF (p80) TNF (p60) receptors, CD27, CD30, CD40, Fas, and NGFreceptor).

Analysis of the zalpha11 sequence suggests that it is a member of thesame receptor subfamily as the IL-2 receptor β-subunit, IL-3, IL-4, andIL-6 receptors. Certain receptors in this subfamily (e.g., G-CSF)associate to form homodimers that transduce a signal. Other members ofthe subfamily (e.g., IL-6, IL-11, and LIF receptors) combine with asecond subunit (termed a β-subunit) to bind ligand and transduce asignal. Specific β-subunits associate with a plurality of specificcytokine receptor subunits. For example, the β-subunit gp130 (Hibi etal., Cell 63:1149-1157, 1990) associates with receptor subunits specificfor IL-6, IL-11, and LIF (Gearing et al., EMBO J. 10:2839-2848, 1991;Gearing et al., U.S. Pat. No. 5,284,755). Oncostatin M binds to aheterodimer of LIF receptor and gp130. CNTF binds to trimeric receptorscomprising CNTF receptor, LIF receptor, and gp130 subunits.

A polynucleotide sequence for the mouse ortholog of human zalpha11 hasbeen identified and is shown in SEQ ID NO:84 and the corresponding aminoacid sequence shown in SEQ ID NO: 85. Analysis of the mouse zalpha11polypeptide encoded by the DNA sequence of SEQ ID NO:84 revealed an openreading frame encoding 529 amino acids (SEQ ID NO:85) comprising apredicted secretory signal peptide of 19 amino acid residues (residue 1(Met) to residue 19 (Ser) of SEQ ID NO:85), and a mature polypeptide of510 amino acids (residue 20 (Cys) to residue 529 (Ser) of SEQ ID NO:2).In addition to the WSXWS motif (SEQ ID NO:3) corresponding to residues214 to 218 of SEQ ID NO:85, the receptor comprises a cytokine-bindingdomain of approximately 200 amino acid residues (residues 20 (Cys) to237 (His) of SEQ ID NO:85); a domain linker (residues 120 (Pro) to 123(Pro) of SEQ ID NO:85); a penultimate strand region (residues 192 (Lys)to 202 (Ala) of SEQ ID NO:85); a transmembrane domain (residues 238(Met) to 254 (Leu) of SEQ ID NO:85); complete intracellular signalingdomain (residues 255 (Lys) to 529 (Ser) of SEQ ID NO:85) which containsa “Box I” signaling site (residues 266 (Ile) to 273 (Pro) of SEQ IDNO:85), and a “Box II” signaling site (residues 301 (Ile) to 304 (Val)of SEQ ID NO:2). A comparison of the human and mouse amino acidsequences reveals that both the human and orthologous polypeptidescontain corresponding structural features described above (See, FIG. 2).The mature sequence for the mouse zalpha11 begins at Cys₂₀ (as shown inSEQ ID NO:85), which corresponds to Cys₂₀ (as shown in SEQ ID NO:2) inthe human sequence. There is about 63% identity between the mouse andhuman sequences over the entire amino acid sequence corresponding to SEQID NO:2 and SEQ ID NO:85. There is about 69% identity a between themouse and human zalpha11 sequences over the extracellular cytokinebinding domain corresponding to residues 20 (Cys) to 237 (His) of SEQ IDNO:2 and residues 20 (Cys) to 237 (His) of SEQ ID NO:85. There is about60% identity a between the mouse and human zalpha11 sequences over theintracellular signalling domain corresponding to residues 256 (Lys) to538 (Ser) of SEQ ID NO:2, and residues 255 (Lys) to 529 (Ser) of SEQ IDNO:85. The above percent identities were determined using a FASTAprogram with ktup=1, gap opening penalty=12, gap extension penalty=2,and substitution matrix=BLOSUM62, with other parameters set as default.The corresponding polynucleotides encoding the mouse zalpha11polypeptide regions, domains, motifs, residues and sequences describedabove are as shown in SEQ ID NO:84.

Those skilled in the art will recognize that the sequence disclosed inSEQ ID NO:1 represents a single allele of human zalpha11 and thatallelic variation and alternative splicing are expected to occur.Allelic variants of this sequence can be cloned by probing cDNA orgenomic libraries from different individuals according to standardprocedures. Allelic variants of the DNA sequence shown in SEQ ID NO:1,including those containing silent mutations and those in which mutationsresult in amino acid sequence changes, are within the scope of thepresent invention, as are proteins which are allelic variants of SEQ IDNO:2. cDNAs generated from alternatively spliced mRNAs, which retain theproperties of the zalpha11 polypeptide are included within the scope ofthe present invention, as are polypeptides encoded by such cDNAs andmRNAs. Allelic variants and splice variants of these sequences can becloned by probing cDNA or genomic libraries from different individualsor tissues according to standard procedures known in the art.

The present invention also provides isolated zalpha11 polypeptides thatare substantially similar to the polypeptides of SEQ ID NO:2 and theirorthologs. The term “substantially similar” is used herein to denotepolypeptides having at least 70%, more preferably at least 80%, sequenceidentity to the sequences shown in SEQ ID NO:2 or their orthologs. Suchpolypeptides will more preferably be at least 90% identical, and mostpreferably 95% or more identical to SEQ ID NO:2 or its orthologs.)Percent sequence identity is determined by conventional methods. See,for example, Altschul et al., Bull. Math. Bio. 48: 603-616, 1986 andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992.Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “blosum 62” scoring matrix of Henikoff and Henikoff (ibid.) asshown in Table 3 (amino acids are indicated by the standard one-lettercodes). The percent identity is then calculated as:

$\frac{{Total}\mspace{14mu}{number}\mspace{14mu}{of}{\mspace{14mu}\;}{identical}\mspace{14mu}{matches}}{\begin{bmatrix}{{length}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{longer}\mspace{14mu}{sequence}\mspace{14mu}{plus}\mspace{14mu}{the}} \\{{number}\mspace{14mu}{of}\mspace{14mu}{gaps}{\mspace{11mu}\;}{introduced}{\mspace{11mu}\;}{into}\mspace{14mu}{the}\mspace{14mu}{longer}} \\{{sequence}\mspace{14mu}{in}\mspace{14mu}{order}\mspace{14mu}{to}\mspace{14mu}{align}\mspace{14mu}{the}\mspace{14mu}{two}\mspace{14mu}{sequences}}\end{bmatrix}} \times 100$

TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2−2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3−2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2−3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3−1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2−2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1−2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4−2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1−1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0−3 −1 4

Sequence identity of polynucleotide molecules is determined by similarmethods using a ratio as disclosed above.

Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “FASTA”similarity search algorithm of Pearson and Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and the amino acid sequence of a putativevariant zsig57. The FASTA algorithm is described by Pearson and Lipman,Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth.Enzmmol. 183:63 (1990).

Briefly, FASTA first characterizes sequence similarity by identifyingregions shared by the query sequence (e.g., SEQ ID NO:2) and a testsequence that have either the highest density of identities (if the ktupvariable is 1) or pairs of identities (if ktup=2), without consideringconservative amino acid substitutions, insertions, or deletions. The tenregions with the highest density of identities are then rescored bycomparing the similarity of all paired amino acids using an amino acidsubstitution matrix, and the ends of the regions are “trimmed” toinclude only those residues that contribute to the highest score. Ifthere are several regions with scores greater than the “cutoff” value(calculated by a predetermined formula based upon the length of thesequence and the ktup value), then the trimmed initial regions areexamined to determine whether the regions can be joined to form anapproximate alignment with gaps. Finally, the highest scoring regions ofthe two amino acid sequences are aligned using a modification of theNeedleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol.48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), which allowsfor amino acid insertions and deletions. Preferred parameters for FASTAanalysis are: ktup=1, gap opening penalty=10, gap extension penalty=1,and substitution matrix=BLOSUM62, with other parameters set as default.These parameters can be introduced into a FASTA program by modifying thescoring matrix file (“SMATRIX”), as explained in Appendix 2 of Pearson,Meth. Enzmmol. 183:63 (1990).

FASTA can also be used to determine the sequence identity of nucleicacid molecules using a ratio as disclosed above. For nucleotide sequencecomparisons, the ktup value can range between one to six, preferablyfrom three to six, most preferably three, with other parameters set asdefault.

The BLOSUM62 table (Table 3) is an amino acid substitution matrixderived from about 2,000 local multiple alignments of protein sequencesegments, representing highly conserved regions of more than 500 groupsof related proteins (Henikoff and Henikoff, Proc. Nat'l Acad. Sci. USA89:10915 (1992)). Accordingly, the BLOSUM62 substitution frequencies canbe used to define conservative amino acid substitutions that may beintroduced into the amino acid sequences of the present invention.Although it is possible to design amino acid substitutions based solelyupon chemical properties (as discussed below), the language“conservative amino acid substitution” preferably refers to asubstitution represented by a BLOSUM62 value of greater than −1. Forexample, an amino acid substitution is conservative if the substitutionis characterized by a BLOSUM62 value of 0, 1, 2, or 3. According to thissystem, preferred conservative amino acid substitutions arecharacterized by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), whilemore preferred conservative amino acid substitutions are characterizedby a BLOSUM62 value of at least 2 (e.g., 2 or 3).

Variant zalpha11 polypeptides or substantially homologous zalpha11polypeptides are characterized as having one or more amino acidsubstitutions, deletions or additions. These changes are preferably of aminor nature, that is conservative amino acid substitutions (see Table4) and other substitutions that do not significantly affect the foldingor activity of the polypeptide; small deletions, typically of one toabout 30 amino acids; and small amino- or carboxyl-terminal extensions,such as an amino-terminal methionine residue, a small linker peptide ofup to about 20-25 residues, or an affinity tag. The present inventionthus includes polypeptides of from about 489 to about 568 amino acidresidues that comprise a sequence that is at least 80%, preferably atleast 90%, and more preferably 95% or more identical to thecorresponding region of SEQ ID NO:2. Polypeptides comprising affinitytags can further comprise a proteolytic cleavage site between thezalpha11 polypeptide and the affinity tag. Suitable sites includethrombin cleavage sites and factor Xa cleavage sites.

TABLE 4 Conservative amino acid substitutions Basic: arginine lysinehistidine Acidic: glutamic acid aspartic acid Polar: glutamineasparagine Hydrophobic: leucine isoleucine valine Aromatic:phenylalanine tryptophan tyrosine Small: glycine alanine serinethreonine methionine

The present invention further provides a variety of other polypeptidefusions and related multimeric proteins comprising one or morepolypeptide fusions. For example, a zalpha11 polypeptide can be preparedas a fusion to a dimerizing protein as disclosed in U.S. Pat. Nos.5,155,027 and 5,567,584. Preferred dimerizing proteins in this regardinclude immunoglobulin constant region domains. Immunoglobulin-zalpha11polypeptide fusions can be expressed in genetically engineered cells toproduce a variety of multimeric zalpha11 analogs. Auxiliary domains canbe fused to zalpha11 polypeptides to target them to specific cells,tissues, or macromolecules (e.g., collagen). A zalpha11 polypeptide canbe fused to two or more moieties, such as an affinity tag forpurification and a targeting domain. Polypeptide fusions can alsocomprise one or more cleavage sites, particularly between domains. See,Tuan et al., Connective Tissue Research 34:1-9, 1996.

The proteins of the present invention can also comprise non-naturallyoccurring amino acid residues. Non-naturally occurring amino acidsinclude, without limitation, trans-3-methylproline, 2,4-methanoproline,cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine,allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline,3,3-dimethylproline, tent-leucine, norvaline, 2-azaphenylalanine,3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.Several methods are known in the art for incorporating non-naturallyoccurring amino acid residues into proteins. For example, an in vitrosystem can be employed wherein nonsense mutations are suppressed usingchemically aminoacylated suppressor tRNAs. Methods for synthesizingamino acids and aminoacylating tRNA are known in the art. Transcriptionand translation of plasmids containing nonsense mutations is carried outin a cell-free system comprising an E. coli S30 extract and commerciallyavailable enzymes and other reagents. Proteins are purified bychromatography. See, for example, Robertson et al., J. Am. Chem. Soc.113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung etal., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci.USA 90:10145-9, 1993). In a second method, translation is carried out inXenopus oocytes by microinjection of mutated mRNA and chemicallyaminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem.271:19991-8, 1996). Within a third method, E. coli cells are cultured inthe absence of a natural amino acid that is to be replaced (e.g.,phenylalanine) and in the presence of the desired non-naturallyoccurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturallyoccurring amino acid is incorporated into the protein in place of itsnatural counterpart. See, Koide et al., Biochem. 33:7470-7476, 1994.Naturally occurring amino acid residues can be converted tonon-naturally occurring species by in vitro chemical modification.Chemical modification can be combined with site-directed mutagenesis tofurther expand the range of substitutions (Wynn and Richards, ProteinSci. 2:395-403, 1993).

A limited number of non-conservative amino acids, amino acids that arenot encoded by the genetic code, non-naturally occurring amino acids,and unnatural amino acids may be substituted for zalpha11 amino acidresidues.

Essential amino acids in the polypeptides of the present invention canbe identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244: 1081-5, 1989; Bass et al., Proc. Natl. Acad.Sci. USA 88:4498-502, 1991). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity (e.g.ligand binding and signal transduction) as disclosed below to identifyamino acid residues that are critical to the activity of the molecule.See also, Hilton et al., J. Biol. Chem. 271:4699-4708, 1996. Sites ofligand-receptor, protein-protein or other biological interaction canalso be determined by physical analysis of structure, as determined bysuch techniques as nuclear magnetic resonance, crystallography, electrondiffraction or photoaffinity labeling, in conjunction with mutation ofputative contact site amino acids. See, for example, de Vos et al.,Science 255:306-312, 1992; Smith et al., J. Mol. Biol. 224:899-904,1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identities ofessential amino acids can also be inferred from analysis of homologieswith related receptors.

Determination of amino acid residues that are within regions or domainsthat are critical to maintaining structural integrity can be determined.Within these regions one can determine specific residues that will bemore or less tolerant of change and maintain the overall tertiarystructure of the molecule. Methods for analyzing sequence structureinclude, but are not limited to, alignment of multiple sequences withhigh amino acid or nucleotide identity and computer analysis usingavailable software (e.g., the INSIGHT II® viewer and homology modelingtools; MSI, San Diego, Calif.), secondary structure propensities, binarypatterns, complementary packing and buried polar interactions (Barton,Current Opin. Struct. Biol. 5:372-376, 1995 and Cordes et al., CurrentOpin. Struct. Biol. 6:3-10, 1996). In general, when designingmodifications to molecules or identifying specific fragmentsdetermination of structure will be accompanied by evaluating activity ofmodified molecules.

Amino acid sequence changes are made in zalpha11 polypeptides so as tominimize disruption of higher order structure essential to biologicalactivity. For example, when the zalpha11 polypeptide comprises one ormore helices, changes in amino acid residues will be made so as not todisrupt the helix geometry and other components of the molecule wherechanges in conformation abate some critical function, for example,binding of the molecule to its binding partners. The effects of aminoacid sequence changes can be predicted by, for example, computermodeling as disclosed above or determined by analysis of crystalstructure (see, e.g., Lapthorn et al., Nat. Struct. Biol. 2:266-268,1995). Other techniques that are well known in the art compare foldingof a variant protein to a standard molecule (e.g., the native protein).For example, comparison of the cysteine pattern in a variant andstandard molecules can be made. Mass spectrometry and chemicalmodification using reduction and alkylation provide methods fordetermining cysteine residues which are associated with disulfide bondsor are free of such associations (Bean et al., Anal. Biochem.201:216-226, 1992; Gray, Protein Sci. 2:1732-1748, 1993; and Pattersonet al., Anal. Chem. 66:3727-3732, 1994). It is generally believed thatif a modified molecule does not have the same disulfide bonding patternas the standard molecule folding would be affected. Another well knownand accepted method for measuring folding is circular dichrosism (CD).Measuring and comparing the CD spectra generated by a modified moleculeand standard molecule is routine (Johnson, Proteins 7:205-214, 1990).Crystallography is another well known method for analyzing folding andstructure. Nuclear magnetic resonance (NMR), digestive peptide mappingand epitope mapping are also known methods for analyzing folding andstructural similarities between proteins and polypeptides (Schaanan etal., Science 257:961-964, 1992).

A Hopp/Woods hydrophilicity profile of the zalpha11 protein sequence asshown in SEQ ID NO:2 can be generated (Hopp et al., Proc. Natl. Acad.Sci. 78:3824-3828, 1981; Hopp, J. Immun. Meth. 88:1-18, 1986 andTriquier et al., Protein Engineering 11:153-169, 1998). See, FIG. 1. Theprofile is based on a sliding six-residue window. Buried G, S, and Tresidues and exposed H, Y, and W residues were ignored. For example, inzalpha11, hydrophilic regions include amino acid residues 55 through 60of SEQ ID NO: 2, amino acid residues 56 through 61 of SEQ ID NO: 2,amino acid residues 139 through 144 of SEQ ID NO: 2, amino acid residues227 through 232 of SEQ ID NO: 2, and amino acid residues 364 through 369of SEQ ID NO: 2.

Those skilled in the art will recognize that hydrophilicity orhydrophobicity will be taken into account when designing modificationsin the amino acid sequence of a zalpha11 polypeptide, so as not todisrupt the overall structural and biological profile. Of particularinterest for replacement are hydrophobic residues selected from thegroup consisting of Val, Leu and Ile or the group consisting of Met,Gly, Ser, Ala, Tyr and Trp. For example, residues tolerant ofsubstitution could include such residues as shown in SEQ ID NO: 2.However, Cysteine residues could be relatively intolerant ofsubstitution.

The identities of essential amino acids can also be inferred fromanalysis of sequence similarity between class I cytokine receptor familymembers with zalpha11. Using methods such as “FASTA” analysis describedpreviously, regions of high similarity are identified within a family ofproteins and used to analyze amino acid sequence for conserved regions.An alternative approach to identifying a variant zalpha11 polynucleotideon the basis of structure is to determine whether a nucleic acidmolecule encoding a potential variant zalpha11 polynucleotide canhybridize to a nucleic acid molecule having the nucleotide sequence ofSEQ ID NO:1, as discussed above.

Other methods of identifying essential amino acids in the polypeptidesof the present invention are procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244:1081 (1989), Bass et al., Proc. Natl. Acad. Sci.USA 88:4498 (1991), Coombs and Corey, “Site-Directed Mutagenesis andProtein Engineering,” in Proteins: Analysis and Design, Angeletti (ed.),pages 259-311 (Academic Press, Inc. 1998)). In the latter technique,single alanine mutations are introduced at every residue in themolecule, and the resultant mutant molecules are tested for biologicalactivity as disclosed below to identify amino acid residues that arecritical to the activity of the molecule. See also, Hilton et al., J.Biol. Chem. 271:4699 (1996).

The present invention also includes functional fragments of zalpha11polypeptides and nucleic acid molecules encoding such functionalfragments. A “functional” zalpha11 or fragment thereof defined herein ischaracterized by its proliferative or differentiating activity, by itsability to induce or inhibit specialized cell functions, or by itsability to bind specifically to an anti-zalpha11 antibody or zalpha11ligand (either soluble or immobilized). As previously described herein,zalpha11 is characterized by a class I cytokine receptor structure.Thus, the present invention further provides fusion proteinsencompassing: (a) polypeptide molecules comprising an extracellular orintracellular domain described herein; and (b) functional fragmentscomprising one or more of these domains. The other polypeptide portionof the fusion protein may be contributed by another class I cytokinereceptor, for exapmple, IL-2 receptor β-subunit and the β-commonreceptor (i.e., IL3, IL-5, and GM-CSF receptor β-subunits), or by anon-native and/or an unrelated secretory signal peptide that facilitatessecretion of the fusion protein.

Routine deletion analyses of nucleic acid molecules can be performed toobtain functional fragments of a nucleic acid molecule that encodes azalpha11 polypeptide. As an illustration, DNA molecules having thenucleotide sequence of SEQ ID NO:1 or fragments thereof, can be digestedwith Bal31 nuclease to obtain a series of nested deletions. These DNAfragments are then inserted into expression vectors in proper readingframe, and the expressed polypeptides are isolated and tested forzalpha11 activity, or for the ability to bind anti-zalpha11 antibodiesor zalpha11 ligand. One alternative to exonuclease digestion is to useoligonucleotide-directed mutagenesis to introduce deletions or stopcodons to specify production of a desired zalpha11 fragment.Alternatively, particular fragments of a zalpha11 polynucleotide can besynthesized using the polymerase chain reaction.

Standard methods for identifying functional domains are well-known tothose of skill in the art. For example, studies on the truncation ateither or both termini of interferons have been summarized byHorisberger and Di Marco, Pharmac. Ther. 66:507 (1995). Moreover,standard techniques for functional analysis of proteins are describedby, for example, Treuter et al., Molec. Gen. Genet. 240:113 (1993);Content et al., “Expression and preliminary deletion analysis of the 42kDa 2-5A synthetase induced by human interferon,” in BiologicalInterferon Systems, Proceedings of ISIR-TNO Meeting on InterferonSystems, Cantell (ed.), pages 65-72 (Nijhoff 1987); Herschman, “The EGFReceptor,” in Control of Animal Cell Proliferation 1, Boynton et al.,(eds.) pages 169-199 (Academic Press 1985); Coumailleau et al., J. Biol.Chem. 270:29270 (1995); Fukunaga et al., J. Biol. Chem. 270:25291(1995); Yamaguchi et al., Biochem. Pharmacol. 50:1295 (1995); and Meiselet al., Plant Molec. Biol. 30:1 (1996).

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53-57, 1988) or Bowie and Sauer(Proc. Natl. Acad. Sci. USA 86:2152-2156, 1989). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832-10837, 1991;Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO92/062045) and region-directed mutagenesis (Derbyshire et al., Gene46:145, 1986; Ner et al., DNA 7:127, 1988).

Variants of the disclosed zalpha11 DNA and polypeptide sequences can begenerated through DNA shuffling as disclosed by Stemmer, Nature370:389-91, 1994, Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-51, 1994and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated byin vitro homologous recombination by random fragmentation of a parentDNA followed by reassembly using PCR, resulting in randomly introducedpoint mutations. This technique can be modified by using a family ofparent DNAs, such as allelic variants or DNAs from different species, tointroduce additional variability into the process. Selection orscreening for the desired activity, followed by additional iterations ofmutagenesis and assay provides for rapid “evolution” of sequences byselecting for desirable mutations while simultaneously selecting againstdetrimental changes.

Mutagenesis methods as disclosed herein can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized zalpha11 receptor polypeptides in host cells.Preferred assays in this regard include cell proliferation assays andbiosensor-based ligand-binding assays, which are described below.Mutagenized DNA molecules that encode active receptors or portionsthereof (e.g., ligand-binding fragments, signaling domains, and thelike) can be recovered from the host cells and rapidly sequenced usingmodern equipment. These methods allow the rapid determination of theimportance of individual amino acid residues in a polypeptide ofinterest, and can be applied to polypeptides of unknown structure.

In addition, the proteins of the present invention (or polypeptidefragments thereof) can be joined to other bioactive molecules,particularly other cytokines, to provide multi-functional molecules. Forexample, one or more helices from zalpha11 can be joined to othercytokines to enhance their biological properties or efficiency ofproduction.

The present invention thus provides a series of novel, hybrid moleculesin which a segment comprising one or more of the helices of zalpha11 isfused to another polypeptide. Fusion is preferably done by splicing atthe DNA level to allow expression of chimeric molecules in recombinantproduction systems. The resultant molecules are then assayed for suchproperties as improved solubility, improved stability, prolongedclearance half-life, improved expression and secretion levels, andpharmacodynamics. Such hybrid molecules may further comprise additionalamino acid residues (e.g. a polypeptide linker) between the componentproteins or polypeptides.

Using the methods discussed herein, one of ordinary skill in the art canidentify and/or prepare a variety of polypeptide fragments or variantsof SEQ ID NO:2 that retain the signal transduction or ligand bindingactivity. For example, one can make a zalpha11 “soluble receptor” bypreparing a variety of polypeptides that are substantially homologous tothe cytokine-binding domain (residues 20 (Cys) to 237 (His) of SEQ IDNO:2 or allelic variants or species orthologs thereof) and retainligand-binding activity of the wild-type zalpha11 protein. Suchpolypeptides may include additional amino acids from, for example, partor all of the transmembrane and intracellular domains. Such polypeptidesmay also include additional polypeptide segments as generally disclosedherein such as labels, affinity tags, and the like.

For any zalpha11 polypeptide, including variants, soluble receptors, andfusion polypeptides or proteins, one of ordinary skill in the art canreadily generate a fully degenerate polynucleotide sequence encodingthat variant using the information set forth in Tables 1 and 2 above.

The zalpha11 polypeptides of the present invention, includingfull-length polypeptides, biologically active fragments, and fusionpolypeptides, can be produced in genetically engineered host cellsaccording to conventional techniques. Suitable host cells are those celltypes that can be transformed or transfected with exogenous DNA andgrown in culture, and include bacteria, fungal cells, and culturedhigher eukaryotic cells. Eukaryotic cells, particularly cultured cellsof multicellular organisms, are preferred. Techniques for manipulatingcloned DNA molecules and introducing exogenous DNA into a variety ofhost cells are disclosed by Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989, and Ausubel et al., eds., Current Protocolsin Molecular Biology, John Wiley and Sons, Inc., NY, 1987.

In general, a DNA sequence encoding a zalpha11 polypeptide is operablylinked to other genetic elements required for its expression, generallyincluding a transcription promoter and terminator, within an expressionvector. The vector will also commonly contain one or more selectablemarkers and one or more origins of replication, although those skilledin the art will recognize that within certain systems selectable markersmay be provided on separate vectors, and replication of the exogenousDNA may be provided by integration into the host cell genome. Selectionof promoters, terminators, selectable markers, vectors and otherelements is a matter of routine design within the level of ordinaryskill in the art. Many such elements are described in the literature andare available through commercial suppliers.

To direct a zalpha11 polypeptide into the secretory pathway of a hostcell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in the expression vector.The secretory signal sequence may be that of zalpha11, or may be derivedfrom another secreted protein (e.g., t-PA) or synthesized de novo. Thesecretory signal sequence is operably linked to the zalpha11 DNAsequence, i.e., the two sequences are joined in the correct readingframe and positioned to direct the newly synthesized polypeptide intothe secretory pathway of the host cell. Secretory signal sequences arecommonly positioned 5′ to the DNA sequence encoding the polypeptide ofinterest, although certain secretory signal sequences may be positionedelsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S.Pat. No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830).

Alternatively, the secretory signal sequence contained in thepolypeptides of the present invention is used to direct otherpolypeptides into the secretory pathway. The present invention providesfor such fusion polypeptides. A signal fusion polypeptide can be madewherein a secretory signal sequence derived from amino acid 1 (Met) toamino acid 19 (Gly) of SEQ ID NO:2 is operably linked to anotherpolypeptide using methods known in the art and disclosed herein. Thesecretory signal sequence contained in the fusion polypeptides of thepresent invention is preferably fused amino-terminally to an additionalpeptide to direct the additional peptide into the secretory pathway.Such constructs have numerous applications known in the art. Forexample, these novel secretory signal sequence fusion constructs candirect the secretion of an active component of a normally non-secretedprotein. Such fusions may be used in vivo or in vitro to direct peptidesthrough the secretory pathway.

Cultured mammalian cells are suitable hosts within the presentinvention. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran mediatedtransfection (Ausubel et al., ibid.), and liposome-mediated transfection(Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80,1993, and viral vectors (Miller and Rosman, BioTechniques 7:980-90,1989; Wang and Finer, Nature Med. 2:714-716, 1996). The production ofrecombinant polypeptides in cultured mammalian cells is disclosed, forexample, by Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al., U.S.Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; andRingold, U.S. Pat. No. 4,656,134. Suitable cultured mammalian cellsinclude the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK(ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamsterovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines. Additional suitablecell lines are known in the art and available from public depositoriessuch as the American Type Culture Collection, Rockville, Md. In general,strong transcription promoters are preferred, such as promoters fromSV-40 or cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Othersuitable promoters include those from metallothionein genes (U.S. Pat.Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.

Drug selection is generally used to select for cultured mammalian cellsinto which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems canalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g. hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used. Alternative markers that introducean altered phenotype, such as green fluorescent protein, or cell surfaceproteins such as CD4, CD8, Class I MHC, placental alkaline phosphatasemay be used to sort transfected cells from untransfected cells by suchmeans as FACS sorting or magnetic bead separation technology.

Other higher eukaryotic cells can also be used as hosts, including plantcells, insect cells and avian cells. The use of Agrobacterium rhizogenesas a vector for expressing genes in plant cells has been reviewed bySinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987. Transformation ofinsect cells and production of foreign polypeptides therein is disclosedby Guarino et al., U.S. Pat. No. 5,162,222 and WIPO publication WO94/06463. Insect cells can be infected with recombinant baculovirus,commonly derived from Autographa californica nuclear polyhedrosis virus(AcNPV). See, King, L. A. and Possee, R. D., The Baculovirus ExpressionSystem: A Laboratory Guide, London, Chapman & Hall; O'Reilly, D. R. etal., Baculovirus Expression Vectors: A Laboratory Manual, New York,Oxford University Press., 1994; and, Richardson, C. D., Ed., BaculovirusExpression Protocols. Methods in Molecular Biology, Totowa, N.J., HumanaPress, 1995. A second method of making recombinant zalpha11 baculovirusutilizes a transposon-based system described by Luckow (Luckow, V. A, etal., J Virol 67:4566-79, 1993). This system, which utilizes transfervectors, is sold in the Bac-to-Bac™ kit (Life Technologies, Rockville,Md.). This system utilizes a transfer vector, pFastBac 1™ (LifeTechnologies) containing a Tn7 transposon to move the DNA encoding thezalpha11 polypeptide into a baculovirus genome maintained in E. coli asa large plasmid called a “bacmid.” See, Hill-Perkins, M. S, and Possee,R. D., J Gen Virol 71:971-6, 1990; Bonning, B. C. et al., J Gen Virol75:1551-6, 1994; and, Chazenbalk, G. D., and Rapoport, B., J Biol Chem270:1543-9, 1995. In addition, transfer vectors can include an in-framefusion with DNA encoding an epitope tag at the C- or N-terminus of theexpressed zalpha11 polypeptide, for example, a Glu-Glu epitope tag(Grussenmeyer, T. et al., Proc. Natl. Acad. Sci. 82:7952-4, 1985). Usinga technique known in the art, a transfer vector containing zalpha11 istransformed into E. Coli, and screened for bacmids which contain aninterrupted lacZ gene indicative of recombinant baculovirus. The bacmidDNA containing the recombinant baculovirus genome is isolated, usingcommon techniques, and used to transfect Spodoptera frugiperda cells,e.g. Sf9 cells. Recombinant virus that expresses zalpha11 issubsequently produced. Recombinant viral stocks are made by methodscommonly used in the art.

The recombinant virus is used to infect host cells, typically a cellline derived from the fall armyworm, Spodoptera frugiperda. See, ingeneral, Glick and Pasternak, Molecular Biotechnology: Principles andApplications of Recombinant DNA, ASM Press, Washington, D.C., 1994.Another suitable cell line is the High FiveO™ cell line (Invitrogen)derived from Trichoplusia ni (U.S. Pat. No. 5,300,435). Commerciallyavailable serum-free media are used to grow and maintain the cells.Suitable media are Sf900 II™ (Life Technologies) or ESF 921™ (ExpressionSystems) for the Sf9 cells; and Ex-cellO405™ (JRH Biosciences, Lenexa,Kans.) or Express FiveO™ (Life Technologies) for the T. ni cells.Procedures used are generally described in available laboratory manuals(King, L. A. and Possee, R. D., ibid.; O'Reilly, D. R. et al., ibid.;Richardson, C. D., ibid.). Subsequent purification of the zalpha11polypeptide from the supernatant can be achieved using methods describedherein.

Fungal cells, including yeast cells, can also be used within the presentinvention. Yeast species of particular interest in this regard includeSaccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.Methods for transforming S. cerevisiae cells with exogenous DNA andproducing recombinant polypeptides therefrom are disclosed by, forexample, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat.No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat.No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformedcells are selected by phenotype determined by the selectable marker,commonly drug resistance or the ability to grow in the absence of aparticular nutrient (e.g., leucine). A preferred vector system for usein Saccharomyces cerevisiae is the POT1 vector system disclosed byKawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformedcells to be selected by growth in glucose-containing media. Suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman etal., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446;5,063,154; 5,139,936 and 4,661,454. Transformation systems for otheryeasts, including Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia methanolica, Pichia guillermondii and Candida maltosaare known in the art. See, for example, Gleeson et al., J. Gen.Microbiol. 132:3459-3465, 1986 and Cregg, U.S. Pat. No. 4,882,279.Aspergillus cells may be utilized according to the methods of McKnightet al., U.S. Pat. No. 4,935,349. Methods for transforming Acremoniumchrysogenum are disclosed by Sumino et al., U.S. Pat. No. 5,162,228.Methods for transforming Neurospora are disclosed by Lambowitz, U.S.Pat. No. 4,486,533.

The use of Pichia methanolica as host for the production of recombinantproteins is disclosed in WIPO Publications WO 97/17450, WO 97/17451, WO98/02536, and WO 98/02565. DNA molecules for use in transforming P.methanolica will commonly be prepared as double-stranded, circularplasmids, which are preferably linearized prior to transformation. Forpolypeptide production in P. methanolica, it is preferred that thepromoter and terminator in the plasmid be that of a P. methanolica gene,such as a P. methanolica alcohol utilization gene (AUG1 or AUG2). Otheruseful promoters include those of the dihydroxyacetone synthase (DHAS),formate dehydrogenase (FMD), and catalase (CAT) genes. To facilitateintegration of the DNA into the host chromosome, it is preferred to havethe entire expression segment of the plasmid flanked at both ends byhost DNA sequences. A preferred selectable marker for use in Pichiamethanolica is a P. methanolica ADE2 gene, which encodesphosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), whichallows ade2 host cells to grow in the absence of adenine. Forlarge-scale, industrial processes where it is desirable to minimize theuse of methanol, it is preferred to use host cells in which bothmethanol utilization genes (AUG1 and AUG2) are deleted. For productionof secreted proteins, host cells deficient in vacuolar protease genes(PEP4 and PRB1) are preferred. Electroporation is used to facilitate theintroduction of a plasmid containing DNA encoding a polypeptide ofinterest into P. methanolica cells. It is preferred to transform P.methanolica cells by electroporation using an exponentially decaying,pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm,preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40milliseconds, most preferably about 20 milliseconds.

Prokaryotic host cells, including strains of the bacteria Escherichiacoli, Bacillus and other genera are also useful host cells within thepresent invention. Techniques for transforming these hosts andexpressing foreign DNA sequences cloned therein are well known in theart (see, e.g., Sambrook et al., ibid.). When expressing a zalpha11polypeptide in bacteria such as E. coli, the polypeptide may be retainedin the cytoplasm, typically as insoluble granules, or may be directed tothe periplasmic space by a bacterial secretion sequence. In the formercase, the cells are lysed, and the granules are recovered and denaturedusing, for example, guanidine isothiocyanate or urea. The denaturedpolypeptide can then be refolded and dimerized by diluting thedenaturant, such as by dialysis against a solution of urea and acombination of reduced and oxidized glutathione, followed by dialysisagainst a buffered saline solution. In the latter case, the polypeptidecan be recovered from the periplasmic space in a soluble and functionalform by disrupting the cells (by, for example, sonication or osmoticshock) to release the contents of the periplasmic space and recoveringthe protein, thereby obviating the need for denaturation and refolding.

Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell. P. methanolicacells are cultured in a medium comprising adequate sources of carbon,nitrogen and trace nutrients at a temperature of about 25° C. to 35° C.Liquid cultures are provided with sufficient aeration by conventionalmeans, such as shaking of small flasks or sparging of fermentors. Apreferred culture medium for P. methanolica is YEPD (2% D-glucose, 2%Bacto™ Peptone (Difco Laboratories, Detroit, Mich.), 1% Bacto™ yeastextract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).

Within one aspect of the present invention, a zalpha11 cytokine receptor(including transmembrane and intracellular domains) is produced by acultured cell, and the cell is used to screen for ligands for thereceptor, including the natural ligand, as well as agonists andantagonists of the natural ligand. To summarize this approach, a cDNA orgene encoding the receptor is combined with other genetic elementsrequired for its expression (e.g., a transcription promoter), and theresulting expression vector is inserted into a host cell. Cells thatexpress the DNA and produce functional receptor are selected and usedwithin a variety of screening systems.

Mammalian cells suitable for use in expressing the novel receptors ofthe present invention and transducing a receptor-mediated signal includecells that express a β-subunit, such as gp130, and cells that co-expressgp130 and LIF receptor (Gearing et al., EMBO J. 10:2839-2848, 1991;Gearing et al., U.S. Pat. No. 5,284,755). In this regard it is generallypreferred to employ a cell that is responsive to other cytokines thatbind to receptors in the same subfamily, such as IL-6 or LIF, becausesuch cells will contain the requisite signal transduction pathway(s).Preferred cells of this type include the human TF-1 cell line (ATCCnumber CRL-2003) and the DA-1 cell line (Branch et al., Blood 69:1782,1987; Broudy et al., Blood 75:1622-1626, 1990). In the alternative,suitable host cells can be engineered to produce a β-subunit or othercellular component needed for the desired cellular response. Forexample, the murine cell line BaF3 (Palacios and Steinmetz, Cell41:727-734, 1985; Mathey-Prevot et al., Mol. Cell. Biol. 6: 4133-4135,1986), a baby hamster kidney (BHK) cell line, or the CTLL-2 cell line(ATCC TIB-214) can be transfected to express the mouse gp130 subunit, ormouse gp130 and LIF receptor, in addition to zalpha11. It is generallypreferred to use a host cell and receptor(s) from the same species,however this approach allows cell lines to be engineered to expressmultiple receptor subunits from any species, thereby overcomingpotential limitations arising from species specificity. In thealternative, species homologs of the human receptor cDNA can be clonedand used within cell lines from the same species, such as a mouse cDNAin the BaF3 cell line. Cell lines that are dependent upon onehematopoietic growth factor, such as IL-3, can thus be engineered tobecome dependent upon a zalpha11 ligand.

Cells expressing functional zalpha11 are used within screening assays. Avariety of suitable assays are known in the art. These assays are basedon the detection of a biological response in the target cell. One suchassay is a cell proliferation assay. Cells are cultured in the presenceor absence of a test compound, and cell proliferation is detected by,for example, measuring incorporation of tritiated thymidine or bycolorimetric assay based on the metabolic breakdown of Alymar Blue™(AccuMed, Chicago, Ill.) or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Mosman, J. Immunol. Meth. 65: 55-63, 1983).An alternative assay format uses cells that are further engineered toexpress a reporter gene. The reporter gene is linked to a promoterelement that is responsive to the receptor-linked pathway, and the assaydetects activation of transcription of the reporter gene. A preferredpromoter element in this regard is a serum response element, or SRE(see, for example, Shaw et al., Cell 56:563-572, 1989). A preferred suchreporter gene is a luciferase gene (de Wet et al., Mol. Cell. Biol.7:725, 1987). Expression of the luciferase gene is detected byluminescence using methods known in the art (e.g., Baumgartner et al.,J. Biol. Chem. 269:19094-29101, 1994; Schenborn and Goiffin, PromegaNotes 41:11, 1993). Luciferase assay kits are commercially availablefrom, for example, Promega Corp., Madison, Wis. Target cell lines ofthis type can be used to screen libraries of chemicals, cell-conditionedculture media, fungal broths, soil samples, water samples, and the like.For example, a bank of cell- or tissue-conditioned media samples can beassayed on a target cell to identify cells that produce ligand. Positivecells are then used to produce a cDNA library in a mammalian cellexpression vector, which is divided into pools, transfected into hostcells, and expressed. Media samples from the transfected cells are thenassayed, with subsequent division of pools, retransfection,subculturing, and re-assay of positive cells to isolate a clonal cellline expressing the ligand. Media samples conditioned by kidney, liver,spleen, thymus, other lymphoid tissues, or T-cells are preferred sourcesof ligand for use in screening procedures.

A natural ligand for zalpha11 can also be identified by mutagenizing acytokine-dependent cell line expressing zalpha11 and culturing it underconditions that select for autocrine growth. See WIPO publication WO95/21930. Within a typical procedure, cells expressing zalpha11 aremutagenized, such as with EMS. The cells are then allowed to recover inthe presence of the required cytokine, then transferred to a culturemedium lacking the cytokine. Surviving cells are screened for theproduction of a ligand for zalpha11, such as by adding soluble(ligand-binding) receptor polypeptide to the culture medium or byassaying conditioned media on wild-type cells and transfected cellsexpressing the zalpha11. Preferred cell lines for use within this methodinclude cells that are transfected to express gp130 or gp130 incombination with LIF receptor. Preferred such host cell lines includetransfected CTLL-2 cells (Gillis and Smith, Nature 268:154-156, 1977)and transfected BaF3 cells.

Moreover, a secretion trap method employing zalpha11 soluble receptorpolypeptide can be used to isolate a zalpha11 ligand (Aldrich, et al,Cell 87: 1161-1169, 1996). A cDNA expression library prepared from aknown or suspected ligand source is transfected into COS-7 cells. ThecDNA library vector generally has an SV40 origin for amplification inCOS-7 cells, and a CMV promoter for high expression. The transfectedCOS-7 cells are grown in a monolayer and then fixed and permeabilized.Tagged or biotin-labeled zalpha11 soluble receptor, described herein, isthen placed in contact with the cell layer and allowed to bind cells inthe monolayer that express an anti-complementary molecule, i.e., azalpha11 ligand. A cell expressing a ligand will thus be bound withreceptor molecules. An anti-tag antibody (anti-Ig for Ig fusions, M2 oranti-FLAG for FLAG-tagged fusions, streptavidin, and the like) which isconjugated with horseradish peroxidase (HRP) is used to visualize thesecells to which the tagged or biotin-labeled zalpha11 soluble receptorhas bound. The HRP catalyzes deposition of a tyramide reagent, forexample, tyramide-FITC. A commercially-available kit can be used forthis detection (for example, Renaissance TSA-Direct™ Kit; NEN LifeScience Products, Boston, Mass.). Cells which express zalpha11 receptorligand will be identified under fluorescence microscopy as green cellsand picked for subsequent cloning of the ligand using procedures forplasmid rescue as outlined in Aldrich, et al, supra., followed bysubsequent rounds of secretion trap assay until single clones areidentified.

As a receptor, the activity of zalpha11 polypeptide can be measured by asilicon-based biosensor microphysiometer which measures theextracellular acidification rate or proton excretion associated withreceptor binding and subsequent physiologic cellular responses. Anexemplary device is the Cytosensor™ Microphysiometer manufactured byMolecular Devices, Sunnyvale, Calif. A variety of cellular responses,such as cell proliferation, ion transport, energy production,inflammatory response, regulatory and receptor activation, and the like,can be measured by this method. See, for example, McConnell, H. M. etal., Science 257:1906-1912, 1992; Pitchford, S. et al., Meth. Enzymol.228:84-108, 1997; Arimilli, S. et al., J. Immunol. Meth. 212:49-59,1998; Van Liefde, I. Et al., Eur. J. Pharmacol. 346:87-95, 1998. Themicrophysiometer can be used for assaying eukaryotic, prokaryotic,adherent or non-adherent cells. By measuring extracellular acidificationchanges in cell media over time, the microphysiometer directly measurescellular responses to various stimuli, including agonists, ligands, orantagonists of the zalpha11 polypeptide. Preferably, themicrophysiometer is used to measure responses of a zalpha11-expressingeukaryotic cell, compared to a control eukaryotic cell that does notexpress zalpha11 polypeptide. Zalpha11-expressing eukaryotic cellscomprise cells into which zalpha11 has been transfected, as describedherein, creating a cell that is responsive to zalpha11-modulatingstimuli, or are cells naturally expressing zalpha11, such aszalpha11-expressing cells derived from lymphoid, spleen, thymus tissueor PBLs. Differences, measured by an increase or decrease inextracellular acidification, in the response of cells expressingzalpha11, relative to a control, are a direct measurement ofzalpha11-modulated cellular responses. Moreover, such zalpha11-modulatedresponses can be assayed under a variety of stimuli. Also, using themicrophysiometer, there is provided a method of identifying agonists andantagonists of zalpha11 polypeptide, comprising providing cellsexpressing a zalpha11 polypeptide, culturing a first portion of thecells in the absence of a test compound, culturing a second portion ofthe cells in the presence of a test compound, and detecting an increaseor a decrease in a cellular response of the second portion of the cellsas compared to the first portion of the cells. Antagonists and agonists,including the natural ligand for zalpha11 polypeptide, can be rapidlyidentified using this method.

Additional assays provided by the present invention include the use ofhybrid receptor polypeptides. These hybrid polypeptides fall into twogeneral classes. Within the first class, the intracellular domain ofzalpha11, comprising approximately residues 256 (Lys) to 528 (Ser) ofSEQ ID NO:2, is joined to the ligand-binding domain of a secondreceptor. It is preferred that the second receptor be a hematopoieticcytokine receptor, such as mpl receptor (Souyri et al., Cell63:1137-1147, 1990). The hybrid receptor will further comprise atransmembrane domain, which may be derived from either receptor. A DNAconstruct encoding the hybrid receptor is then inserted into a hostcell. Cells expressing the hybrid receptor are cultured in the presenceof a ligand for the binding domain and assayed for a response. Thissystem provides a means for analyzing signal transduction mediated byzalpha11 while using readily available ligands. This system can also beused to determine if particular cell lines are capable of responding tosignals transduced by zalpha11. A second class of hybrid receptorpolypeptides comprise the extracellular (ligand-binding) domain ofzalpha11 (approximately residues 20 (Cys) to 237 (His) of SEQ ID NO:2)with a cytoplasmic domain of a second receptor, preferably a cytokinereceptor, and a transmembrane domain. The transmembrane domain may bederived from either receptor. Hybrid receptors of this second class areexpressed in cells known to be capable of responding to signalstransduced by the second receptor. Together, these two classes of hybridreceptors enable the use of a broad spectrum of cell types withinreceptor-based assay systems.

Cells found to express a ligand for zalpha11 are then used to prepare acDNA library from which the ligand-encoding cDNA may be isolated asdisclosed above. The present invention thus provides, in addition tonovel receptor polypeptides, methods for cloning polypeptide ligands forthe receptors.

The tissue specificity of zalpha11 expression suggests a role in earlythymocyte development and immune response regulation. These processesinvolve stimulation of cell proliferation and differentiation inresponse to the binding of one or more cytokines to their cognatereceptors. In view of the tissue distribution observed for thisreceptor, agonists (including the natural ligand) and antagonists haveenormous potential in both in vitro and in vivo applications. Compoundsidentified as receptor agonists are useful for stimulating proliferationand development of target cells in vitro and in vivo. For example,agonist compounds are useful as components of defined cell culturemedia, and may be used alone or in combination with other cytokines andhormones to replace serum that is commonly used in cell culture.Agonists are thus useful in specifically promoting the growth and/ordevelopment of T-cells, B-cells, and other cells of the lymphoid andmyeloid lineages, and hematopoietic cells in culture.

Agonist ligands for zalpha11 may be useful in stimulating cell-mediatedimmunity and for stimulating lymphocyte proliferation, such as in thetreatment of infections involving immunosuppression, including certainviral infections. Additional uses include tumor suppression, wheremalignant transformation results in tumor cells that are antigenic.Agonist ligands could be used to induce cytotoxicity, which may bemediated through activation of effector cells such as T-cells, NK(natural killer) cells, or LAK (lymphoid activated killer) cells, orinduced directly through apoptotic pathways. Agonist ligands may also beuseful in treating leukopenias by increasing the levels of the affectedcell type, and for enhancing the regeneration of the T-cell repertoireafter bone marrow transplantation.

Antagonist ligands or compounds may find utility in the suppression ofthe immune system, such as in the treatment of autoimmune diseases,including rheumatoid arthritis, multiple sclerosis, diabetes mellitis,inflammatory bowel disease, Crohn's disease, etc. Immune suppression canalso be used to reduce rejection of tissue or organ transplants andgrafts and to treat T-cell specific leukemias or lymphomas by inhibitingproliferation of the affected cell type.

Zalpha11 may also be used within diagnostic systems for the detection ofcirculating levels of ligand. Within a related embodiment, antibodies orother agents that specifically bind to zalpha11 can be used to detectcirculating receptor polypeptides. Elevated or depressed levels ofligand or receptor polypeptides may be indicative of pathologicalconditions, including cancer. Soluble receptor polypeptides maycontribute to pathologic processes and can be an indirect marker of anunderlying disease. For example, elevated levels of soluble IL-2receptor in human serum have been associated with a wide variety ofinflammatory and neoplastic conditions, such as myocardial infarction,asthma, myasthenia gravis, rheumatoid arthritis, acute T-cell leukemia,B-cell lymphomas, chronic lymphocytic leukemia, colon cancer, breastcancer, and ovarian cancer (Heaney et al., Blood 87:847-857, 1996).

A ligand-binding polypeptide of a zalpha11 receptor, or “solublereceptor,” can be prepared by expressing a truncated DNA encoding thezalpha11 cytokine binding domain (approximately residue 20 (Cys) throughresidue 237 (His) of the human receptor (SEQ ID NO:2)) or thecorresponding region of a non-human receptor. It is preferred that theextracellular domain be prepared in a form substantially free oftransmembrane and intracellular polypeptide segments. Moreover,ligand-binding polypeptide fragments within the zalpha11 cytokinebinding domain, described above, can also serve as zalpha11 solublereceptors for uses described herein. To direct the export of a receptorpolypeptide from the host cell, the receptor DNA is linked to a secondDNA segment encoding a secretory peptide, such as a t-PA secretorypeptide or a zalpha11 secretory peptide. To facilitate purification ofthe secreted receptor polypeptide, a C-terminal extension, such as apoly-histidine tag, substance P, Flag™ peptide (Hopp et al.,Bio/Technology 6:1204-1210, 1988; available from Eastman Kodak Co., NewHaven, Conn.) or another polypeptide or protein for which an antibody orother specific binding agent is available, can be fused to the receptorpolypeptide.

In an alternative approach, a receptor extracellular domain can beexpressed as a fusion with immunoglobulin heavy chain constant regions,typically an F_(c) fragment, which contains two constant region domainsand lacks the variable region. Such fusions are typically secreted asmultimeric molecules wherein the Fc portions are disulfide bonded toeach other and two receptor polypeptides are arrayed in close proximityto each other. Fusions of this type can be used to affinity purify thecognate ligand from solution, as an in vitro assay tool, to blocksignals in vitro by specifically titrating out ligand, and asantagonists in vivo by administering them parenterally to bindcirculating ligand and clear it from the circulation. To purify ligand,a zalpha11-Ig chimera is added to a sample containing the ligand (e.g.,cell-conditioned culture media or tissue extracts) under conditions thatfacilitate receptor-ligand binding (typically near-physiologicaltemperature, pH, and ionic strength). The chimera-ligand complex is thenseparated by the mixture using protein A, which is immobilized on asolid support (e.g., insoluble resin beads). The ligand is then elutedusing conventional chemical techniques, such as with a salt or pHgradient. In the alternative, the chimera itself can be bound to a solidsupport, with binding and elution carried out as above. Collectedfractions can be re-fractionated until the desired level of purity isreached.

Moreover, zalpha11 soluble receptors can be used as a “ligand sink,”i.e., antagonist, to bind ligand in vivo or in vitro in therapeutic orother applications where the presence of the ligand is not desired. Forexample, in cancers that are expressing large amount of bioactivezalpha11 ligand, zalpha11 soluble receptors can be used as a directantagonist of the ligand in vivo, and may aid in reducing progressionand symptoms associated with the disease. Moreover, zalpha11 solublereceptor can be used to slow the progression of cancers thatover-express zalkpha11 receptors, by binding ligand in vivo that wouldotherwise enhance proliferation of those cancers. Similar in vitroapplications for a zalpha11 soluble receptor can be used, for instance,as a negative selection to select cell lines that grow in the absence ofzalpha11 ligand.

Moreover, zalpha11 soluble receptor can be used in vivo or in diagnosticapplications to detect zalpha11 ligand-expressing cancers in vivo or intissue samples. For example, the zalpha11 soluble receptor can beconjugated to a radio-label or fluorescent label as described herein,and used to detect the presence of the ligand in a tissue sample usingan in vitro ligand-receptor type binding assay, or fluorescent imagingassay. Moreover, a radio-labeled zalpha11 soluble receptor could beadministered in vivo to detect ligand-expressing solid tumors through aradio-imaging method known in the art.

Analysis of the tissue distribution of the mRNA corresponding to thisnovel DNA showed expression in lymphoid tissues, including thymus,spleen, lymph nodes, and peripheral blood leukocytes. These dataindicate a role for the zalpha11 receptor in proliferation,differentiation, and/or activation of immune cells, and suggest a rolein development and regulation of immune responses. The data also suggestthat the interaction of zalpha11 with its ligand may stimulateproliferation and development of myeloid cells and may, like IL-2, IL-6,LIF, IL-11 and OSM (Baumann et al., J. Biol. Chem. 268:8414-8417, 1993),induce acute-phase protein synthesis in hepatocytes.

It is preferred to purify the polypeptides of the present invention to≧80% purity, more preferably to ≧90% purity, even more preferably ≧95%purity, and particularly preferred is a pharmaceutically pure state,that is greater than 99.9% pure with respect to contaminatingmacromolecules, particularly other proteins and nucleic acids, and freeof infectious and pyrogenic agents. Preferably, a purified polypeptideis substantially free of other polypeptides, particularly otherpolypeptides of animal origin.

Expressed recombinant zalpha11 polypeptides (or zalpha11 chimeric orfusion polypeptides) can be purified using fractionation and/orconventional purification methods and media. Ammonium sulfateprecipitation and acid or chaotrope extraction may be used forfractionation of samples. Exemplary purification steps may includehydroxyapatite, size exclusion, FPLC and reverse-phase high performanceliquid chromatography. Suitable chromatographic media includederivatized dextrans, agarose, cellulose, polyacrylamide, specialtysilicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred.Exemplary chromatographic media include those media derivatized withphenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia),Toyopearl butyl 650 (Toso Haas, Montgomeryville, Pa.), Octyl-Sepharose(Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG71 (Toso Haas) and the like. Suitable solid supports include glassbeads, silica-based resins, cellulosic resins, agarose beads,cross-linked agarose beads, polystyrene beads, cross-linkedpolyacrylamide resins and the like that are insoluble under theconditions in which they are to be used. These supports may be modifiedwith reactive groups that allow attachment of proteins by amino groups,carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydratemoieties. Examples of coupling chemistries include cyanogen bromideactivation, N-hydroxysuccinimide activation, epoxide activation,sulfhydryl activation, hydrazide activation, and carboxyl and aminoderivatives for carbodiimide coupling chemistries. These and other solidmedia are well known and widely used in the art, and are available fromcommercial suppliers. Methods for binding receptor polypeptides tosupport media are well known in the art. Selection of a particularmethod is a matter of routine design and is determined in part by theproperties of the chosen support. See, for example, AffinityChromatography: Principles & Methods, Pharmacia LKB Biotechnology,Uppsala, Sweden, 1988.

The polypeptides of the present invention can be isolated byexploitation of their biochemical, structural, and biologicalproperties. For example, immobilized metal ion adsorption (IMAC)chromatography can be used to purify histidine-rich proteins, includingthose comprising polyhistidine tags. Briefly, a gel is first chargedwith divalent metal ions to form a chelate (Sulkowski, Trends inBiochem. 3:1-7, 1985). Histidine-rich proteins will be adsorbed to thismatrix with differing affinities, depending upon the metal ion used, andwill be eluted by competitive elution, lowering the pH, or use of strongchelating agents. Other methods of purification include purification ofglycosylated proteins by lectin affinity chromatography and ion exchangechromatography (Methods in Enzymol., Vol. 182, “Guide to ProteinPurification”, M. Deutscher, (ed.), Acad. Press, San Diego, 1990, pp.529-39). Within additional embodiments of the invention, a fusion of thepolypeptide of interest and an affinity tag (e.g., maltose-bindingprotein, an immunoglobulin domain) may be constructed to facilitatepurification.

Moreover, using methods described in the art, polypeptide fusions, orhybrid zalpha11 proteins, are constructed using regions or domains ofthe inventive zalpha11 in combination with those of other human cytokinereceptor family proteins, or heterologous proteins (Sambrook et al.,ibid., Altschul et al., ibid., Picard, Cur. Opin. Biology, 5:511-5,1994, and references therein). These methods allow the determination ofthe biological importance of larger domains or regions in a polypeptideof interest. Such hybrids may alter reaction kinetics, binding,constrict or expand the substrate specificity, or alter tissue andcellular localization of a polypeptide, and can be applied topolypeptides of unknown structure.

Fusion polypeptides or proteins can be prepared by methods known tothose skilled in the art by preparing each component of the fusionprotein and chemically conjugating them. Alternatively, a polynucleotideencoding one or more components of the fusion protein in the properreading frame can be generated using known techniques and expressed bythe methods described herein. For example, part or all of a domain(s)conferring a biological function may be swapped between zalpha11 of thepresent invention with the functionally equivalent domain(s) fromanother cytokine family member. Such domains include, but are notlimited to, the secretory signal sequence, extracellular cytokinebinding domain, transmembrane domain, and intracellular signalingdomain, Box I and Box II sites, as disclosed herein. Such fusionproteins would be expected to have a biological functional profile thatis the same or similar to polypeptides of the present invention or otherknown family proteins, depending on the fusion constructed. Moreover,such fusion proteins may exhibit other properties as disclosed herein.

Standard molecular biological and cloning techniques can be used to swapthe equivalent domains between the zalpha11 polypeptide and thosepolypeptides to which they are fused. Generally, a DNA segment thatencodes a domain of interest, e.g., a zalpha11 domain described herein,is operably linked in frame to at least one other DNA segment encodingan additional polypeptide (for instance a domain or region from anothercytokine receptor, such as the IL-2 receptor), and inserted into anappropriate expression vector, as described herein. Generally DNAconstructs are made such that the several DNA segments that encode thecorresponding regions of a polypeptide are operably linked in frame tomake a single construct that encodes the entire fusion protein, or afunctional portion thereof. For example, a DNA construct would encodefrom N-terminus to C-terminus a fusion protein comprising a signalpolypeptide followed by a cytokine binding domain, followed by atransmembrane domain, followed by an intracellular signaling domain.Such fusion proteins can be expressed, isolated, and assayed foractivity as described herein.

Zalpha11 polypeptides or fragments thereof may also be prepared throughchemical synthesis. zalpha11 polypeptides may be monomers or multimers;glycosylated or non-glycosylated; pegylated or non-pegylated; and may ormay not include an initial methionine amino acid residue.

Polypeptides of the present invention can also be synthesized byexclusive solid phase synthesis, partial solid phase methods, fragmentcondensation or classical solution synthesis. Methods for synthesizingpolypeptides are well known in the art. See, for example, Merrifield, J.Am. Chem. Soc. 85:2149, 1963; Kaiser et al., Anal. Biochem. 34:595,1970. After the entire synthesis of the desired peptide on a solidsupport, the peptide-resin is with a reagent which cleaves thepolypeptide from the resin and removes most of the side-chain protectinggroups. Such methods are well established in the art.

The activity of molecules of the present invention can be measured usinga variety of assays that measure cell differentiation and proliferation.Such assays are well known in the art.

Proteins of the present invention are useful for example, in treatinglymphoid, immune, inflammatory, spleenic, blood or bone disorders, andcan be measured in vitro using cultured cells or in vivo byadministering molecules of the claimed invention to the appropriateanimal model. For instance, host cells expressing a zalpha11 solublereceptor polypeptide can be embedded in an alginate environment andinjected (implanted) into recipient animals. Alginate-poly-L-lysinemicroencapsulation, permselective membrane encapsulation and diffusionchambers are a means to entrap transfected mammalian cells or primarymammalian cells. These types of non-immunogenic “encapsulations” permitthe diffusion of proteins and other macromolecules secreted or releasedby the captured cells to the recipient animal. Most importantly, thecapsules mask and shield the foreign, embedded cells from the recipientanimal's immune response. Such encapsulations can extend the life of theinjected cells from a few hours or days (naked cells) to several weeks(embedded cells). Alginate threads provide a simple and quick means forgenerating embedded cells.

The materials needed to generate the alginate threads are known in theart. In an exemplary procedure, 3% alginate is prepared in sterile H₂O,and sterile filtered. Just prior to preparation of alginate threads, thealginate solution is again filtered. An approximately 50% cellsuspension (containing about 5×10⁵ to about 5×10⁷ cells/ml) is mixedwith the 3% alginate solution. One ml of the alginate/cell suspension isextruded into a 100 mM sterile filtered CaCl₂ solution over a timeperiod of ˜15 min, forming a “thread”. The extruded thread is thentransferred into a solution of 50 mM CaCl₂, and then into a solution of25 mM CaCl₂. The thread is then rinsed with deionized water beforecoating the thread by incubating in a 0.01% solution of poly-L-lysine.Finally, the thread is rinsed with Lactated Ringer's Solution and drawnfrom solution into a syringe barrel (without needle). A large boreneedle is then attached to the syringe, and the thread isintraperitoneally injected into a recipient in a minimal volume of theLactated Ringer's Solution.

An in vivo approach for assaying proteins of the present inventioninvolves viral delivery systems. Exemplary viruses for this purposeinclude adenovirus, herpesvirus, retroviruses, vaccinia virus, andadeno-associated virus (AAV). Adenovirus, a double-stranded DNA virus,is currently the best studied gene transfer vector for delivery ofheterologous nucleic acid (for review, see T. C. Becker et al., Meth.Cell Biol. 43:161-89, 1994; and J. T. Douglas and D. T. Curiel, Science& Medicine 4:44-53, 1997). The adenovirus system offers severaladvantages: (i) adenovirus can accommodate relatively large DNA inserts;(ii) can be grown to high-titer; (iii) infect a broad range of mammaliancell types; and (iv) can be used with a large number of differentpromoters including ubiquitous, tissue specific, and regulatablepromoters. Also, because adenoviruses are stable in the bloodstream,they can be administered by intravenous injection.

Using adenovirus vectors where portions of the adenovirus genome aredeleted, inserts are incorporated into the viral DNA by direct ligationor by homologous recombination with a co-transfected plasmid. In anexemplary system, the essential E1 gene has been deleted from the viralvector, and the virus will not replicate unless the E1 gene is providedby the host cell (the human 293 cell line is exemplary). Whenintravenously administered to intact animals, adenovirus primarilytargets the liver. If the adenoviral delivery system has an E1 genedeletion, the virus cannot replicate in the host cells. However, thehost's tissue (e.g., liver) will express and process (and, if asecretory signal sequence is present, secrete) the heterologous protein.Secreted proteins will enter the circulation in the highly vascularizedliver, and effects on the infected animal can be determined.

Moreover, adenoviral vectors containing various deletions of viral genescan be used in an attempt to reduce or eliminate immune responses to thevector. Such adenoviruses are E1 deleted, and in addition containdeletions of E2A or E4 (Lusky, M. et al., J. Virol. 72:2022-2032, 1998;Raper, S. E. et al., Human Gene Therapy 9:671-679, 1998). In addition,deletion of E2b is reported to reduce immune responses (Amalfitano, A.et al., J. Virol. 72:926-933, 1998). Moreover, by deleting the entireadenovirus genome, very large inserts of heterologous DNA can beaccommodated. Generation of so called “gutless” adenoviruses where allviral genes are deleted are particularly advantageous for insertion oflarge inserts of heterologous DNA. For review, see Yeh, P. andPerricaudet, M., FASEB J. 11:615-623, 1997.

The adenovirus system can also be used for protein production in vitro.By culturing adenovirus-infected non-293 cells under conditions wherethe cells are not rapidly dividing, the cells can produce proteins forextended periods of time. For instance, BHK cells are grown toconfluence in cell factories, then exposed to the adenoviral vectorencoding the secreted protein of interest. The cells are then grownunder serum-free conditions, which allows infected cells to survive forseveral weeks without significant cell division. Alternatively,adenovirus vector infected 293 cells can be grown as adherent cells orin suspension culture at relatively high cell density to producesignificant amounts of protein (See Garnier et al., Cytotechnol.15:145-55, 1994). With either protocol, an expressed, secretedheterologous protein can be repeatedly isolated from the cell culturesupernatant, lysate, or membrane fractions depending on the dispositionof the expressed protein in the cell. Within the infected 293 cellproduction protocol, non-secreted proteins may also be effectivelyobtained.

In view of the tissue distribution observed for zalpha11, agonists(including the natural ligand/substrate/cofactor/etc.) and antagonistshave enormous potential in both in vitro and in vivo applications.Compounds identified as zalpha11 agonists are useful for stimulatinggrowth of immune and hematopoietic cells in vitro and in vivo. Forexample, zalpha11 and agonist compounds are useful as components ofdefined cell culture media, and may be used alone or in combination withother cytokines and hormones to replace serum that is commonly used incell culture. Agonists are thus useful in specifically promoting thegrowth and/or development of T-cells, B-cells, and other cells of thelymphoid and myeloid lineages in culture. Moreover, zalpha11 solublereceptor, agonist, or antagonist may be used in vitro in an assay tomeasure stimulation of colony formation from isolated primary bonemarrow cultures. Such assays are well known in the art.

Antagonists are also useful as research reagents for characterizingsites of ligand-receptor interaction. Inhibitors of zalpha11 activity(zalpha11 antagonists) include anti-zalpha11 antibodies and solublezalpha11 receptors, as well as other peptidic and non-peptidic agents(including ribozymes).

Zalpha11 can also be used to identify modulators (e.g, antagonists) ofits activity. Test compounds are added to the assays disclosed herein toidentify compounds that inhibit the activity of zalpha11. In addition tothose assays disclosed herein, samples can be tested for inhibition ofzalpha11 activity within a variety of assays designed to measurezalpha11 binding, oligomerization, or the stimulation/inhibition ofzalpha11-dependent cellular responses. For example, zalpha11-expressingcell lines can be transfected with a reporter gene construct that isresponsive to a zalpha11-stimulated cellular pathway. Reporter geneconstructs of this type are known in the art, and will generallycomprise a zalpha11-DNA response element operably linked to a geneencoding an assay detectable protein, such as luciferase. DNA responseelements can include, but are not limited to, cyclic AMP responseelements (CRE), hormone response elements (HRE) insulin response element(IRE) (Nasrin et al., Proc. Natl. Acad. Sci. USA 87:5273-7, 1990) andserum response elements (SRE) (Shaw et al. Cell 56: 563-72, 1989).Cyclic AMP response elements are reviewed in Roestler et al., J. Biol.Chem. 263 (19):9063-6; 1988 and Habener, Molec. Endocrinol. 4(8):1087-94; 1990. Hormone response elements are reviewed in Beato, Cell56:335-44; 1989. Candidate compounds, solutions, mixtures or extracts orconditioned media from various cell types are tested for the ability toenhance the activity of zalpha11 receptor as evidenced by a increase inzalpha11 stimulation of reporter gene expression. Assays of this typewill detect compounds that directly stimulate zalpha11 signaltransduction activity through binding the receptor or by otherwisestimulating part of the signal cascade. As such, there is provided amethod of identifying agonists of zalpha11 polypeptide, comprisingproviding cells responsive to a zalpha11 polypeptide, culturing a firstportion of the cells in the absence of a test compound, culturing asecond portion of the cells in the presence of a test compound, anddetecting a increase in a cellular response of the second portion of thecells as compared to the first portion of the cells. Moreover thirdcell, containing the reporter gene construct described above, but notexpressing zalpha11 receptor, can be used as a control cell to assessnon-specific, or non-zalpha11-mediated, stimulation of the reporter.Agonists, including the natural ligand, are therefore useful tostimulate or increase zalpha11 polypeptide function.

A zalpha11 ligand-binding polypeptide, such as the cytokine bindingdomain disclosed herein, can also be used for purification of ligand.The polypeptide is immobilized on a solid support, such as beads ofagarose, cross-linked agarose, glass, cellulosic resins, silica-basedresins, polystyrene, cross-linked polyacrylamide, or like materials thatare stable under the conditions of use. Methods for linking polypeptidesto solid supports are known in the art, and include amine chemistry,cyanogen bromide activation, N-hydroxysuccinimide activation, epoxideactivation, sulfhydryl activation, and hydrazide activation. Theresulting medium will generally be configured in the form of a column,and fluids containing ligand are passed through the column one or moretimes to allow ligand to bind to the receptor polypeptide. The ligand isthen eluted using changes in salt concentration, chaotropic agents(guanidine HCl), or pH to disrupt ligand-receptor binding.

An assay system that uses a ligand-binding receptor (or an antibody, onemember of a complement/anti-complement pair) or a binding fragmentthereof, and a commercially available biosensor instrument may beadvantageously employed (e.g., BIAcore™, Pharmacia Biosensor,Piscataway, N.J.; or SELDI™ technology, Ciphergen, Inc., Palo Alto,Calif.). Such receptor, antibody, member of a complement/anti-complementpair or fragment is immobilized onto the surface of a receptor chip. Useof this instrument is disclosed by Karlsson, J. Immunol. Methods145:229-240, 1991 and Cunningham and Wells, J. Mol. Biol. 234:554-63,1993. A receptor, antibody, member or fragment is covalently attached,using amine or sulfhydryl chemistry, to dextran fibers that are attachedto gold film within the flow cell. A test sample is passed through thecell. If a ligand, epitope, or opposite member of thecomplement/anti-complement pair is present in the sample, it will bindto the immobilized receptor, antibody or member, respectively, causing achange in the refractive index of the medium, which is detected as achange in surface plasmon resonance of the gold film. This system allowsthe determination of on- and off-rates, from which binding affinity canbe calculated, and assessment of stoichiometry of binding.

Ligand-binding receptor polypeptides can also be used within other assaysystems known in the art. Such systems include Scatchard analysis fordetermination of binding affinity (see Scatchard, Ann. NY Acad. Sci. 51:660-672, 1949) and calorimetric assays (Cunningham et al., Science253:545-48, 1991; Cunningham et al., Science 245:821-25, 1991).

Zalpha11 polypeptides can also be used to prepare antibodies that bindto zalpha11 epitopes, peptides or polypeptides. The zalpha11 polypeptideor a fragment thereof serves as an antigen (immunogen) to inoculate ananimal and elicit an immune response. One of skill in the art wouldrecognize that antigens or immunogenic epitopes can consist of stretchesof amino acids within a longer polypeptide, from about 10 amino acidsand up to about the entire length of the polypeptide or longer dependingon the polypeptide. Suitable antigens include the zalpha11 polypeptideencoded by SEQ ID NO:2 from amino acid number 20 (Cys) to amino acidnumber 538 (Ser), or a contiguous 9 to 519 AA amino acid fragmentthereof. Preferred peptides to use as antigens are the cytokine bindingdomain, intracellular signaling domain, Box I and Box II sites,disclosed herein, and zalpha11 hydrophilic peptides such as thosepredicted by one of skill in the art from a hydrophobicity plot,determined for example, from a Hopp/Woods hydrophilicity profile basedon a sliding six-residue window, with buried G, S, and T residues andexposed H, Y, and W residues ignored (See, FIG. 1). Zalpha11 hydrophilicpeptides include peptides comprising amino acid sequences selected fromthe group consisting of: (1) amino acid number 51 (Trp) to amino acidnumber 61 (Glu) of SEQ ID NO:2; (2) amino acid number 136 (Ile) to aminoacid number 143 (Glu) of SEQ ID NO:2; (3) amino acid number 187 (Pro) toamino acid number 195 (Ser) of SEQ ID NO:2; (4) amino acid number 223(Phe) to amino acid number 232 (Glu) of SEQ ID NO:2; and (5) amino acidnumber 360 (Glu) to amino acid number 368 (Asp) of SEQ ID NO:2. Inaddition, conserved motifs, and variable regions between conservedmotifs of zalpha11 are suitable antigens. Moreover, correspondingregions of the mouse zalpha11 polypeptide (SEQ ID NO:85) can be used togenerate antibodies against the mouse zalpha11. Antibodies generatedfrom this immune response can be isolated and purified as describedherein. Methods for preparing and isolating polyclonal and monoclonalantibodies are well known in the art. See, for example, CurrentProtocols in Immunology, Cooligan, et al. (eds.), National Institutes ofHealth, John Wiley and Sons, Inc., 1995; Sambrook et al., MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y.,1989; and Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies:Techniques and Applications, CRC Press, Inc., Boca Raton, Fla., 1982.

As would be evident to one of ordinary skill in the art, polyclonalantibodies can be generated from inoculating a variety of warm-bloodedanimals such as horses, cows, goats, sheep, dogs, chickens, rabbits,mice, and rats with a zalpha11 polypeptide or a fragment thereof. Theimmunogenicity of a zalpha11 polypeptide may be increased through theuse of an adjuvant, such as alum (aluminum hydroxide) or Freund'scomplete or incomplete adjuvant. Polypeptides useful for immunizationalso include fusion polypeptides, such as fusions of zalpha11 or aportion thereof with an immunoglobulin polypeptide or with maltosebinding protein. The polypeptide immunogen may be a full-length moleculeor a portion thereof. If the polypeptide portion is “hapten-like”, suchportion may be advantageously joined or linked to a macromolecularcarrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin(BSA) or tetanus toxoid) for immunization.

As used herein, the term “antibodies” includes polyclonal antibodies,affinity-purified polyclonal antibodies, monoclonal antibodies, andantigen-binding fragments, such as F(ab′)₂ and Fab proteolyticfragments. Genetically engineered intact antibodies or fragments, suchas chimeric antibodies, Fv fragments, single chain antibodies and thelike, as well as synthetic antigen-binding peptides and polypeptides,are also included. Non-human antibodies may be humanized by graftingnon-human CDRs onto human framework and constant regions, or byincorporating the entire non-human variable domains (optionally“cloaking” them with a human-like surface by replacement of exposedresidues, wherein the result is a “veneered” antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans is reduced.

Alternative techniques for generating or selecting antibodies usefulherein include in vitro exposure of lymphocytes to zalpha11 protein orpeptide, and selection of antibody display libraries in phage or similarvectors (for instance, through use of immobilized or labeled zalpha11protein or peptide). Genes encoding polypeptides having potentialzalpha11 polypeptide binding domains can be obtained by screening randompeptide libraries displayed on phage (phage display) or on bacteria,such as E. coli. Nucleotide sequences encoding the polypeptides can beobtained in a number of ways, such as through random mutagenesis andrandom polynucleotide synthesis. These random peptide display librariescan be used to screen for peptides which interact with a known targetwhich can be a protein or polypeptide, such as a ligand or receptor, abiological or synthetic macromolecule, or organic or inorganicsubstances. Techniques for creating and screening such random peptidedisplay libraries are known in the art (Ladner et al., U.S. Pat. No.5,223,409; Ladner et al., U.S. Pat. No. 4,946,778; Ladner et al., U.S.Pat. No. 5,403,484 and Ladner et al., U.S. Pat. No. 5,571,698) andrandom peptide display libraries and kits for screening such librariesare available commercially, for instance from Clontech (Palo Alto,Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc.(Beverly, Mass.) and Pharmacia LKB Biotechnology Inc. (Piscataway,N.J.). Random peptide display libraries can be screened using thezalpha11 sequences disclosed herein to identify proteins which bind tozalpha11. These “binding peptides” which interact with zalpha11polypeptides can be used for tagging cells; for isolating homologpolypeptides by affinity purification; they can be directly orindirectly conjugated to drugs, toxins, radionuclides and the like.These binding peptides can also be used in analytical methods such asfor screening expression libraries and neutralizing activity. Thebinding peptides can also be used for diagnostic assays for determiningcirculating levels of zalpha11 polypeptides; for detecting orquantitating soluble zalpha11 polypeptides as marker of underlyingpathology or disease. These binding peptides can also act as zalpha11“antagonists” to block zalpha11 binding and signal transduction in vitroand in vivo. These anti-zalpha11 binding peptides would be useful forinhibiting the action of a ligand that binds with zalpha11.

Antibodies are determined to be specifically binding if: 1) they exhibita threshold level of binding activity, and/or 2) they do notsignificantly cross-react with related polypeptide molecules. First,antibodies herein specifically bind if they bind if they bind to azalpha11 polypeptide, peptide or epitope with an affinity at least10-fold greater than the binding affinity to control (non-zalpha11)polypeptide. It is preferred that the antibodies exhibit a bindingaffinity (K_(a)) of 10⁶ M⁻¹ or greater, preferably 10⁷ M⁻¹ or greater,more preferably 10⁸ M⁻¹ or greater, and most preferably 10⁹ M⁻¹ orgreater. The binding affinity of an antibody can be readily determinedby one of ordinary skill in the art, for example, by Scatchard analysis(Scatchard, G., Ann NY Acad. Sci. 51: 660-672, 1949).

Second, antibodies are determined to specifically bind if they do notsignificantly cross-react with related polypeptides. Antibodies do notsignificantly cross-react with related polypeptide molecules, forexample, if they detect zalpha11 but not known related polypeptidesusing a standard Western blot analysis (Ausubel et al., ibid.). Examplesof known related polypeptides are orthologs, proteins from the samespecies that are members of a protein family (e.g. IL-6), zalpha11polypeptides, and non-human zalpha11. Moreover, antibodies may be“screened against” known related polypeptides to isolate a populationthat specifically binds to the inventive polypeptides. For example,antibodies raised to zalpha11 are adsorbed to related polypeptidesadhered to insoluble matrix; antibodies specific to zalpha11 will flowthrough the matrix under the proper buffer conditions. Such screeningallows isolation of polyclonal and monoclonal antibodiesnon-crossreactive to closely related polypeptides (Antibodies: ALaboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor LaboratoryPress, 1988; Current Protocols in Immunology, Cooligan, et al. (eds.),National Institutes of Health, John Wiley and Sons, Inc., 1995).Screening and isolation of specific antibodies is well known in the art.See, Fundamental Immunology, Paul (eds.), Raven Press, 1993; Getzoff etal., Adv. in Immunol. 43: 1-98, 1988; Monoclonal Antibodies: Principlesand Practice, Goding, J. W. (eds.), Academic Press Ltd., 1996; Benjaminet al., Ann. Rev. Immunol 2: 67-101, 1984.

A variety of assays known to those skilled in the art can be utilized todetect antibodies which specifically bind to zalpha11 proteins orpeptides. Exemplary assays are described in detail in Antibodies: ALaboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor LaboratoryPress, 1988. Representative examples of such assays include: concurrentimmunoelectrophoresis, radioimmunoassay, radioimmuno-precipitation,enzyme-linked immunosorbent assay (ELISA), dot blot or Western blotassay, inhibition or competition assay, and sandwich assay. In addition,antibodies can be screened for binding to wild-type versus mutantzalpha11 protein or polypeptide.

Antibodies to zalpha11 may be used for tagging cells that expresszalpha11; for isolating zalpha11 by affinity purification; fordiagnostic assays for determining circulating levels of zalpha11polypeptides; for detecting or quantitating soluble zalpha11 as markerof underlying pathology or disease; in analytical methods employingFACS; for screening expression libraries; for generating anti-idiotypicantibodies; and as neutralizing antibodies or as antagonists to blockzalpha11 activity in vitro and in vivo. Suitable direct tags or labelsinclude radionuclides, enzymes, substrates, cofactors, inhibitors,fluorescent markers, chemiluminescent markers, magnetic particles andthe like; indirect tags or labels may feature use of biotin-avidin orother complement/anti-complement pairs as intermediates. Antibodiesherein may also be directly or indirectly conjugated to drugs, toxins,radionuclides and the like, and these conjugates used for in vivodiagnostic or therapeutic applications. Moreover, antibodies to zalpha11or fragments thereof may be used in vitro to detect denatured zalpha11or fragments thereof in assays, for example, Western Blots or otherassays known in the art.

Antibodies to zalpha11 are useful for tagging cells that express thereceptor and assaying Zalpha11 expression levels, for affinitypurification, within diagnostic assays for determining circulatinglevels of soluble receptor polypeptides, analytical methods employingfluorescence-activated cell sorting. Divalent antibodies may be used asagonists to mimic the effect of the zalpha11 ligand.

Antibodies herein can also be directly or indirectly conjugated todrugs, toxins, radionuclides and the like, and these conjugates used forin vivo diagnostic or therapeutic applications. For instance, antibodiesor binding polypeptides which recognize zalpha11 of the presentinvention can be used to identify or treat tissues or organs thatexpress a corresponding anti-complementary molecule (i.e., a zalpha11receptor). More specifically, anti-zalpha11 antibodies, or bioactivefragments or portions thereof, can be coupled to detectable or cytotoxicmolecules and delivered to a mammal having cells, tissues or organs thatexpress the zalpha11 molecule.

Suitable detectable molecules may be directly or indirectly attached topolypeptides that bind zalpha11 (“binding polypeptides,” includingbinding peptides disclosed above), antibodies, or bioactive fragments orportions thereof. Suitable detectable molecules include radionuclides,enzymes, substrates, cofactors, inhibitors, fluorescent markers,chemiluminescent markers, magnetic particles and the like. Suitablecytotoxic molecules may be directly or indirectly attached to thepolypeptide or antibody, and include bacterial or plant toxins (forinstance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and thelike), as well as therapeutic radionuclides, such as iodine-131,rhenium-188 or yttrium-90 (either directly attached to the polypeptideor antibody, or indirectly attached through means of a chelating moiety,for instance). Binding polypeptides or antibodies may also be conjugatedto cytotoxic drugs, such as adriamycin. For indirect attachment of adetectable or cytotoxic molecule, the detectable or cytotoxic moleculecan be conjugated with a member of a complementary/anticomplementarypair, where the other member is bound to the binding polypeptide orantibody portion. For these purposes, biotin/streptavidin is anexemplary complementary/anticomplementary pair.

In another embodiment, binding polypeptide-toxin fusion proteins orantibody-toxin fusion proteins can be used for targeted cell or tissueinhibition or ablation (for instance, to treat cancer cells or tissues).Alternatively, if the binding polypeptide has multiple functionaldomains (i.e., an activation domain or a ligand binding domain, plus atargeting domain), a fusion protein including only the targeting domainmay be suitable for directing a detectable molecule, a cytotoxicmolecule or a complementary molecule to a cell or tissue type ofinterest. In instances where the fusion protein including only a singledomain includes a complementary molecule, the anti-complementarymolecule can be conjugated to a detectable or cytotoxic molecule. Suchdomain-complementary molecule fusion proteins thus represent a generictargeting vehicle for cell/tissue-specific delivery of genericanti-complementary-detectable/cytotoxic molecule conjugates.

In another embodiment, zalpha11 binding polypeptide-cytokine orantibody-cytokine fusion proteins can be used for enhancing in vivokilling of target tissues (for example, blood, lymphoid, colon, and bonemarrow cancers), if the binding polypeptide-cytokine or anti-zalpha11antibody targets the hyperproliferative cell (See, generally, Hornick etal., Blood 89:4437-47, 1997). They described fusion proteins enabletargeting of a cytokine to a desired site of action, thereby providingan elevated local concentration of cytokine. Suitable anti-zalpha11antibodies target an undesirable cell or tissue (i.e., a tumor or aleukemia), and the fused cytokine mediates improved target cell lysis byeffector cells. Suitable cytokines for this purpose include interleukin2 and granulocyte-macrophage colony-stimulating factor (GM-CSF), forinstance.

Alternatively, zalpha11 binding polypeptide or antibody fusion proteinsdescribed herein can be used for enhancing in vivo killing of targettissues by directly stimulating a zalpha11-modulated apoptotic pathway,resulting in cell death of hyperproliferative cells expressing zalpha11.

The bioactive binding polypeptide or antibody conjugates describedherein can be delivered orally, intravenously, intraarterially orintraductally, or may be introduced locally at the intended site ofaction.

Four-helix bundle cytokines that bind to cytokine receptors as well asother proteins produced by activated lymphocytes play an importantbiological role in cell differentiation, activation, recruitment andhomeostasis of cells throughout the body. Therapeutic utility includestreatment of diseases which require immune regulation includingautoimmune diseases, such as, rheumatoid arthritis, multiple sclerosis,myasthenia gravis, systemic lupus erythomatosis and diabetes. Zalpha11antagonists or agonists, including soluble receptors and the naturalligand, may be important in the regulation of inflammation, andtherefore would be useful in treating rheumatoid arthritis, asthma,ulcerative colitis, inflammatory bowel disease, Crohn's disease, andsepsis. There may be a role of zalpha11 antagonists or agonists,including soluble receptors and the natural ligand, in mediatingtumorgenesis, and therefore would be useful in the treatment of cancer.Zalpha11 antagonists or agonists, including soluble receptors and thenatural ligand, may be a potential therapeutic in suppressing the immunesystem which would be important for reducing graft rejection. Zalpha11Ligand may have usefulness in prevention of graft vs. host disease.

Alternatively, zalpha11 antagonists or agonists, including solublereceptors and the natural ligand may activate the immune system whichwould be important in boosting immunity to infectious diseases, treatingimmunocompromised patients, such as HIV+ patient, or in improvingvaccines. In particular, zalpha11 antagonists or agonists, includingsoluble receptors and the natural ligand can modulate, stimulate orexpand NK cells, or their progenitors, and would provide therapeuticvalue in treatment of viral infection, and as an anti-neoplastic factor.NK cells are thought to play a major role in elimination of metastatictumor cells and patients with both metastases and solid tumors havedecreased levels of NK cell activity (Whiteside et. al., Curr. Top.Microbiol. Immunol 230:221-244, 1998).

Polynucleotides encoding zalpha11 polypeptides are useful within genetherapy applications where it is desired to increase or inhibit zalpha11activity. If a mammal has a mutated or absent zalpha11 gene, thezalpha11 gene can be introduced into the cells of the mammal. In oneembodiment, a gene encoding a zalpha11 polypeptide is introduced in vivoin a viral vector. Such vectors include an attenuated or defective DNAvirus, such as, but not limited to, herpes simplex virus (HSV),papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associatedvirus (AAV), and the like. Defective viruses, which entirely or almostentirely lack viral genes, are preferred. A defective virus is notinfective after introduction into a cell. Use of defective viral vectorsallows for administration to cells in a specific, localized area,without concern that the vector can infect other cells. Examples ofparticular vectors include, but are not limited to, a defective herpessimplex virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci.2:320-30, 1991); an attenuated adenovirus vector, such as the vectordescribed by Stratford-Perricaudet et al., J. Clin. Invest. 90:626-30,1992; and a defective adeno-associated virus vector (Samulski et al., J.Virol. 61:3096-101, 1987; Samulski et al., J. Virol. 63:3822-8, 1989).

In another embodiment, a zalpha11 gene can be introduced in a retroviralvector, e.g., as described in Anderson et al., U.S. Pat. No. 5,399,346;Mann et al. Cell 33:153, 1983; Temin et al., U.S. Pat. No. 4,650,764;Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J. Virol.62:1120, 1988; Temin et al., U.S. Pat. No. 5,124,263; InternationalPatent Publication No. WO 95/07358, published Mar. 16, 1995 by Doughertyet al.; and Kuo et al., Blood 82:845, 1993. Alternatively, the vectorcan be introduced by lipofection in vivo using liposomes. Syntheticcationic lipids can be used to prepare liposomes for in vivotransfection of a gene encoding a marker (Feigner et al., Proc. Natl.Acad. Sci. USA 84:7413-7, 1987; Mackey et al., Proc. Natl. Acad. Sci.USA 85:8027-31, 1988). The use of lipofection to introduce exogenousgenes into specific organs in vivo has certain practical advantages.Molecular targeting of liposomes to specific cells represents one areaof benefit. More particularly, directing transfection to particularcells represents one area of benefit. For instance, directingtransfection to particular cell types would be particularly advantageousin a tissue with cellular heterogeneity, such as the pancreas, liver,kidney, and brain. Lipids may be chemically coupled to other moleculesfor the purpose of targeting. Targeted peptides (e.g., hormones orneurotransmitters), proteins such as antibodies, or non-peptidemolecules can be coupled to liposomes chemically.

It is possible to remove the target cells from the body; to introducethe vector as a naked DNA plasmid; and then to re-implant thetransformed cells into the body. Naked DNA vectors for gene therapy canbe introduced into the desired host cells by methods known in the art,e.g., transfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, use of a gene gunor use of a DNA vector transporter. See, e.g., Wu et al., J. Biol. Chem.267:963-7, 1992; Wu et al., J. Biol. Chem. 263:14621-4, 1988.

Antisense methodology can be used to inhibit zalpha11 genetranscription, such as to inhibit cell proliferation in vivo.Polynucleotides that are complementary to a segment of azalpha11-encoding polynucleotide (e.g., a polynucleotide as set froth inSEQ ID NO:1) are designed to bind to zalpha11-encoding mRNA and toinhibit translation of such mRNA. Such antisense polynucleotides areused to inhibit expression of zalpha11 polypeptide-encoding genes incell culture or in a subject.

In addition, as a cell surface molecule, zalpha11 polypeptide can beused as a target to introduce gene therapy into a cell. This applicationwould be particularly appropriate for introducing therapeutic genes intocells in which zalpha11 is normally expressed, such as lymphoid tissueand PBLs, or cancer cells which express zalpha11 polypeptide. Forexample, viral gene therapy, such as described above, can be targeted tospecific cell types in which express a cellular receptor, such aszalpha11 polypeptide, rather than the viral receptor. Antibodies, orother molecules that recognize zalpha11 molecules on the target cell'ssurface can be used to direct the virus to infect and administer genetherapeutic material to that target cell. See, Woo, S. L. C, NatureBiotech. 14:1538, 1996; Wickham, T. J. et al, Nature Biotech.14:1570-1573, 1996; Douglas, J. T et al., Nature Biotech. 14:1574-1578,1996; Rihova, B., Crit. Rev. Biotechnol. 17:149-169, 1997; and Vile, R.G. et al., Mol. Med. Today 4:84-92, 1998. For example, a bispecificantibody containing a virus-neutralizing Fab fragment coupled to azalpha11-specific antibody can be used to direct the virus to cellsexpressing the zalpha11 receptor and allow efficient entry of the viruscontaining a genetic element into the cells. See, for example, Wickham,T. J., et al., J. Virol. 71:7663-7669, 1997; and Wickham, T. J., et al.,J. Virol. 70:6831-6838, 1996.

The present invention also provides reagents which will find use indiagnostic applications. For example, the zalpha11 gene, a probecomprising zalpha11 DNA or RNA or a subsequence thereof can be used todetermine if the zalpha11 gene is present on chromosome 16 or if amutation has occurred. Zalpha11 is located at the 16p11.1 region ofchromosome 16 (See, Example 3). Detectable chromosomal aberrations atthe zalpha11 gene locus include, but are not limited to, aneuploidy,gene copy number changes, insertions, deletions, restriction sitechanges and rearrangements. Such aberrations can be detected usingpolynucleotides of the present invention by employing molecular genetictechniques, such as restriction fragment length polymorphism (RFLP)analysis, fluorescence in situ hybridization methods, short tandemrepeat (STR) analysis employing PCR techniques, and other geneticlinkage analysis techniques known in the art (Sambrook et al., ibid.;Ausubel et. al., ibid.; Marian, Chest 108:255-65, 1995).

The precise knowledge of a gene's position can be useful for a number ofpurposes, including: 1) determining if a sequence is part of an existingcontig and obtaining additional surrounding genetic sequences in variousforms, such as YACs, BACs or cDNA clones; 2) providing a possiblecandidate gene for an inheritable disease which shows linkage to thesame chromosomal region; and 3) cross-referencing model organisms, suchas mouse, which may aid in determining what function a particular genemight have.

The zalpha11 gene is located at the 16p11.1 region of chromosome 16.Several genes of known function map to this region. For example, theinterleukin 4 (IL-4) cytokine receptor alpha-subunit, a member of thehematopoietin receptor family, maps to 16p12.1-p11.2. This subunit mayform a heterodimer with zalpha11. Moreover, zalpha11 polynucleotideprobes can be used to detect abnormalities or genotypes associated withdefects in IL-4 receptor, such as those that are implicated in someallergic inflammatory disorders and asthma (Deichman, K. A. et al., Exp.Allergy 28:151-155; 1998; Mitsuyasu, H. et al., Nature Genet.19:119-120, 1998). In addition, zalpha11 polynucleotide probes can beused to detect abnormalities or genotypes associated with inflammatorybowel disease, where a susceptibility marker maps to 16p12-q13 (Cho, J.H. et al, Proc. Nat. Acad. Sci. 95:7502-7507, 1998). Further, zalpha11polynucleotide probes can be used to detect abnormalities or genotypesassociated with hemoglobin loci located at 16pter-p13.3, andparticularly hemoglobin-alpha defects associated with alpha-thalassemiasyndromes, such as hydrops fetalis (for review, see Chui, M. P., andWaye, J. S. Blood 91:2213-2222, 1998). Moreover, amongst other geneticloci, those for Wilms tumor, type III (16q), Rubenstein-Taybi syndrome(16p13.3), severe infantile polycystic kidney disease (16p13.3), allmanifest themselves in human disease states as well as map to thisregion of the human genome. See the Online Mendellian Inheritance of Man(OMIM) gene map, and references therein, for this region of chromosome16 on a publicly available WWW server. All of these serve as possiblecandidate genes for an inheritable disease which show linkage to thesame chromosomal region as the zalpha11 gene.

Similarly, defects in the zalpha11 locus itself may result in aheritable human disease state. Molecules of the present invention, suchas the polypeptides, antagonists, agonists, polynucleotides andantibodies of the present invention would aid in the detection,diagnosis prevention, and treatment associated with a zalpha11 geneticdefect.

Mice engineered to express the zalpha11 gene, referred to as “transgenicmice,” and mice that exhibit a complete absence of zalpha11 genefunction, referred to as “knockout mice,” may also be generated(Snouwaert et al., Science 257:1083, 1992; Lowell et al., Nature366:740-42, 1993; Capecchi, M. R., Science 244: 1288-1292, 1989;Palmiter, R. D. et al. Annu Rev Genet. 20: 465-499, 1986). For example,transgenic mice that over-express zalpha11, either ubiquitously or undera tissue-specific or tissue-restricted promoter can be used to askwhether over-expression causes a phenotype. For example, over-expressionof a wild-type zalpha11 polypeptide, polypeptide fragment or a mutantthereof may alter normal cellular processes, resulting in a phenotypethat identifies a tissue in which zalpha11 expression is functionallyrelevant and may indicate a therapeutic target for the zalpha11, itsagonists or antagonists. For example, a preferred transgenic mouse toengineer is one that expresses a “dominant-negative” phenotype, such asone that over-expresses the zalpha11 extracellular cytokine bindingdomain with the transmembrane domain attached (approximately amino acids20 (Cys) to 255 (Leu) of SEQ ID NO:2). Moreover, such over-expressionmay result in a phenotype that shows similarity with human diseases.Similarly, knockout zalpha11 mice can be used to determine wherezalpha11 is absolutely required in vivo. The phenotype of knockout miceis predictive of the in vivo effects of that a zalpha11 antagonist, suchas those described herein, may have. The mouse or the human zalpha11cDNA can be used to isolate murine zalpha11 mRNA, cDNA and genomic DNA,which are subsequently used to generate knockout mice. These mice may beemployed to study the zalpha11 gene and the protein encoded thereby inan in vivo system, and can be used as in vivo models for correspondinghuman diseases. Moreover, transgenic mice expression of zalpha11antisense polynucleotides or ribozymes directed against zalpha11,described herein, can be used analogously to transgenic mice describedabove.

For pharmaceutical use, the soluble receptor polypeptides of the presentinvention are formulated for parenteral, particularly intravenous orsubcutaneous, delivery according to conventional methods. Intravenousadministration will be by bolus injection or infusion over a typicalperiod of one to several hours. In general, pharmaceutical formulationswill include a zalpha11 soluble receptor polypeptide in combination witha pharmaceutically acceptable vehicle, such as saline, buffered saline,5% dextrose in water or the like. Formulations may further include oneor more excipients, preservatives, solubilizers, buffering agents,albumin to prevent protein loss on vial surfaces, etc. Methods offormulation are well known in the art and are disclosed, for example, inRemington: The Science and Practice of Pharmacy, Gennaro, ed., MackPublishing Co., Easton, Pa., 19th ed., 1995. Therapeutic doses willgenerally be in the range of 0.1 to 100 μg/kg of patient weight per day,preferably 0.5-20 mg/kg per day, with the exact dose determined by theclinician according to accepted standards, taking into account thenature and severity of the condition to be treated, patient traits, etc.Determination of dose is within the level of ordinary skill in the art.The proteins may be administered for acute treatment, over one week orless, often over a period of one to three days or may be used in chronictreatment, over several months or years. In general, a therapeuticallyeffective amount of zalpha11 soluble receptor polypeptide is an amountsufficient to produce a clinically significant effect.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1 Identification of Human Zalpha11 Using an ESTSequence to Obtain Full-length Zalpha11

Scanning of a translated DNA database resulted in identification of anexpressed sequence tag (EST) sequence found to be a member of the ClassI Cytokine Receptor family and designated zalpha11.

Confirmation of the EST sequence was made by sequence analyses of thecDNA from which the EST originated. This cDNA clone was obtained andsequenced using the following primers: ZC 447 (SEQ ID NO:5), ZC 976 (SEQID NO:6), ZC 19345 (SEQ ID NO:7), ZC 19346 (SEQ ID NO:8), ZC 19349 (SEQID NO:9), and ZC 19350 (SEQ ID NO:10), ZC 19458 (SEQ ID NO:11), ZC 19459(SEQ ID NO:12), ZC 19460 (SEQ ID NO:13), ZC 19461 (SEQ ID NO:14), ZC19572 (SEQ ID NO:15), ZC 19573 (SEQ ID NO:16), ZC 19657 (SEQ ID NO:17).The insert was 2945 bp, and was full-length.

Example 2 Tissue Distribution

Northern blot analysis was performed using Human Multiple TissueNorthern™ Blots (MTN I, MTN II, and MTN III) (Clontech). The cDNAdescribed in Example 1 was used in a PCR reaction using oligos ZC19,181(SEQ ID NO:18) and ZC19,182 (SEQ ID NO:19) as primers. PCR conditionswere as follows: 94° C. for 1.5 minutes; 35 cycles at 94° C. for 15seconds then 68° C. for 30 seconds; 72° C. for 10 minutes; 4° C.overnight A sample of the PCR reaction product was run on a 1.5% agarosegel. A band of the expected size of 175 bp was seen. The 175 bp PCRfragment, was gel purified using a commercially available kit (QiaexII™;Qiagen) and then radioactively labeled with ³²P-dCTP using Rediprime II™(Amersham), a random prime labeling system, according to themanufacturer's specifications. The probe was then purified using aNuc-Trap™ column (Stratagene) according to the manufacturer'sinstructions. ExpressHyb™ (Clontech) solution was used forprehybridization and as a hybridizing solution for the Northern blots.Hybridization took place overnight at 65° C. using 1-2×10⁶ cpm/ml oflabeled probe. The blots were then washed 4 times for 15 minutes in2×SSC/1% SDS at 25° C., followed by a wash in 0.1×SSC/0.1% SDS at 50° C.Transcripts of approximately 3 kb and 5 kb were detected in lymph node,peripheral blood leukocytes, and thymus.

Dot Blots were also performed using Human RNA Master Blots™ (Clontech).The methods and conditions for the Dot Blots are the same as for theMultiple Tissue Blots described above. Dot blot had strongest signals inthymus, lymph node, and spleen.

Northern analysis was also performed using Human Cancer Cell Line MTN™(Clontech). The cDNA described in Example 1 was used in a PCR reactionusing oligos ZC19,907 (SEQ ID NO:20) and ZC19,908 (SEQ ID NO:21) asprimers. PCR conditions were as follows: 35 cycles at 95° C. for 1minute, then 60° C. for 1 minute; 72° C. for 1.5 minutes; 72° C. for 10minutes; 4° C. overnight A sample of the PCR reaction product was run ona 1.5% agarose gel. A band of the expected size of 1.2 kb was seen. The1.2 kb PCR fragment, was gel purified using a commercially available kit(QIAQUICK™ Gel Extraction Kit; Qiagen) and then radioactively labeledwith ³²P-dCTP using Prime-It II™ (Stratagene), a random prime labelingsystem, according to the manufacturer's specifications. The probe wasthen purified using a NUC-TRAPT™ column (Stratagene) according to themanufacturer's instructions. EXPRESSHYB™ (Clontech) solution was usedfor prehybridization and as a hybridizing solution for the Northernblots. Hybridization took place for 2 hours at 65° C. using 1-2×10⁶cpm/ml of labeled probe. The blots were then washed 4 times for 15minutes in 2×SSC/1% SDS at 25° C., followed by two 30 minute washes in0.1×SSC/0.1% SDS at 50° C. A strong signal was seen in the Raji cellline derived from Burkitt's lymphoma.

Example 3 PCR-Based Chromosomal Mapping of the Zalpha11 Gene

Zalpha11 was mapped to chromosome 16 using the commercially available“GeneBridge 4 Radiation Hybrid Panel” (Research Genetics, Inc.,Huntsville, Ala.). The GeneBridge 4 Radiation Hybrid Panel containsPCRable DNAs from each of 93 radiation hybrid clones, plus two controlDNAs (the HFL donor and the A23 recipient). A publicly available WWWserver allows mapping relative to the Whitehead Institute/MIT Center forGenome Research's radiation hybrid map of the human genome (the “WICGR”radiation hybrid map) which was constructed with the GeneBridge 4Radiation Hybrid Panel.

For the mapping of Zalpha11 with the “GeneBridge 4 RH Panel,” 20 μlreactions were set up in a 96-well microtiter plate (Stratagene, LaJolla, Calif.) and used in a “RoboCycler Gradient 96” thermal cycler(Stratagene). Each of the 95 PCR reactions consisted of 2 μl 10×PCRreaction buffer (Clontech Laboratories, Inc., Palo Alto, Calif.), 1.6 μldNTPs mix (2.5 mM each, Perkin-Elmer, Foster City, Calif.), 1 μl senseprimer, ZC 19,954, (SEQ ID NO:22), 1 μl antisense primer, ZC 19,955 (SEQID NO:23), 2 μl “RediLoad” (Research Genetics, Inc., Huntsville, Ala.),0.4 μl 50× ADVANTAGE™ KlenTaq Polymerase Mix (Clontech), 25 ng of DNAfrom an individual hybrid clone or control and ddH2O for a total volumeof 20 μl. The reactions were overlaid with an equal amount of mineraloil and sealed. The PCR cycler conditions were as follows: an initial 1cycle 4 minute denaturation at 94° C.; 35 cycles of a 45 seconds at 94°C., 45 seconds at 68° C., and 1 minute at 72° C.; followed by 7 minutesat 72° C. The reactions were separated by electrophoresis on a 2%agarose gel (Life Technologies).

The results showed that zalpha11 maps 9.54 cR_(—)3000 from the frameworkmarker WI-3768 on the chromosome 16 WICGR radiation hybrid map. Proximaland distal framework markers were WI-3768 and TIGR-A002K05,respectively. The use of surrounding markers positions Zalpha11 in the16p11.1 region on the integrated LDB chromosome 16 map (The GeneticLocation Database, University of Southhampton.

Example 4 Construction of Human MPL-zalpha11 Polypeptide Chimera: MPLExtracellular and TM Domain Fused to the Zalpha11 IntracellularSignaling Domain

The extracellular and transmembrane domains of the MPL receptor wereisolated from a plasmid containing the MPL receptor (PHZ1/MPL plasmid)using PCR with primers ZC17,212 (SEQ ID NO:24) and ZC19,914 (SEQ IDNO:25). The reaction conditions were as follows: 95° C. for 1 min.; 35cycles at 95° C. for 1 min., 45° C. for 1 min., 72° C. for 2 min.;followed by 72° C. at 10 min.; then a 10° C. soak. The PCR product wasrun on a 1% low melting point agarose (Boerhinger Mannheim,Indianapolis, Ind.) and the approximately 1.5 kb MPL receptor fragmentisolated using QIAQUICK™ gel extraction kit (Qiagen) as permanufacturer's instructions.

The intracellular domains of zalpha11 were isolated from a plasmidcontaining zalpha11 receptor cDNA using PCR with primers ZC19,913 (SEQID NO:26) and ZC20,097 (SEQ ID NO:27). The polynucleotide sequencecorresponding to the zalpha11 receptor coding sequence is shown in SEQID NO:1 from nucleotide 69 to 1682. The reaction conditions were as perabove. The PCR product was run on a 1% low melting point agarose(Boerhinger Mannheim) and the approximately 900 bp zalpha11 fragmentisolated using QIAQUICK™ gel extraction kit as per manufacturer'sinstructions.

Each of the isolated fragments described above were mixed at a 1:1volumetric ratio and used in a PCR reaction using ZC17,212 (SEQ IDNO:24) and ZC20,097 (SEQ ID NO:27) to create the MPL-zalpha11 chimera.The reaction conditions were as follows: 95° C. for 1 min.; 35 cycles at95° C. for 1 min., 55° C. for 1 min., 72° C. for 2 min; followed by 72°C. at 10 min.; then a 10° C. soak. The entire PCR product was run on a1% low melting point agarose (Boehringer Mannheim) and the approximately2.4 kb MPL-zalpha11 chimera fragment isolated using QIAQUICK™ gelextraction kit (Qiagen) as per manufacturer's instructions. TheMPL-zalpha11 chimera fragment was digested with EcoRI (BRL) and XbaI(Boerhinger Mannheim) as per manufacturer's instructions. The entiredigest was run on a 1% low melting point agarose (Boehringer Mannheim)and the cleaved MPL-zalpha11 chimera isolated using QIAQUICK™ gelextraction kit (Qiagen) as per manufacturer's instructions. Theresultant cleaved MPL-zalpha11 chimera was inserted into an expressionvector as described below.

Recipient expression vector pZP-5N was digested with EcoRI (BRL) andHindIII (BRL) as per manufacturer's instructions, and gel purified asdescribed above. This vector fragment was combined with the EcoRI andXbaI cleaved MPL-zalpha11 chimera isolated above and a XbaI/HindIIIlinker fragment in a ligation reaction. The ligation was run using T4Ligase (BRL), at 15° C. overnight. A sample of the ligation waselectroporated in to DH10B ELECTROMAX™ electrocompetent E. coli cells(25 μF, 200Ω, 2.3V). Transformants were plated on LB+Ampicillin platesand single colonies screened by PCR to check for the MPL-zalpha11chimera using ZC17,212 (SEQ ID NO:24) and ZC20,097 (SEQ ID NO:27) usingthe PCR conditions as described above.

Confirmation of the MPL-zalpha11 chimera sequence was made by sequenceanalyses using the following primers: ZC12,700 (SEQ ID NO:28), ZC5,020(SEQ ID NO:29), ZC6,675 (SEQ ID NO:30), ZC7,727 (SEQ ID NO:31), ZC8,290(SEQ ID NO:32), ZC19,572 (SEQ ID NO:15), ZC6,622 (SEQ ID NO:33), ZC7,736(SEQ ID NO:34), and ZC9,273 (SEQ ID NO:35). The insert was approximately2.4 bp, and was full-length.

Example 5 MPL-zalpha11 Chimera Based Proliferation in BAF3 Assay UsingALAMAR BLUE™

A. Construction of BaF3 Cells Expressing MPL-zalpha11 Chimera

BaF3, an interleukin-3 (IL-3) dependent pre-lymphoid cell line derivedfrom murine bone marrow (Palacios and Steinmetz, Cell 41: 727-734, 1985;Mathey-Prevot et al., Mol. Cell. Biol. 6: 4133-4135, 1986), wasmaintained in complete media (RPMI medium (JRH Bioscience Inc., Lenexa,Kans.) supplemented with 10% heat-inactivated fetal calf serum, 2 ng/mlmurine IL-3 (mIL-3) (R & D, Minneapolis, Minn.), 2 mM L-GLUTAMAX™ media(Gibco BRL), 1 mM Sodium Pyruvate (Gibco BRL), and PSN antibiotics(GIBCO BRL)). Prior to electroporation, pZP-5N/MPL-zalpha11 DNA (Example4) was prepared and purified using a Qiagen Maxi Prep kit (Qiagen) asper manufacturer's instructions. BaF3 cells for electroporation werewashed once in RPMI media and then resuspended in RPMI media at a celldensity of 10⁷ cells/ml. One ml of resuspended BaF3 cells was mixed with30 μg of the pZP-5N/MPL-zalpha11 plasmid DNA and transferred to separatedisposable electroporation chambers (GIBCO BRL). Following a 15 minuteincubation at room temperature the cells were given two serial shocks(800 lFad/300 V.; 1180 lFad/300 V.) delivered by an electroporationapparatus (CELL-PORATOR™; GIBCO BRL). After a 5 minute recovery time,the electroporated cells were transferred to 50 ml of complete media andplaced in an incubator for 15-24 hours (37° C., 5% CO₂). The cells werethen spun down and resuspended in 50 ml of complete media containingGENETICIN™ (Gibco) selection (500 μg/ml G418) in a T-162 flask toisolate the G418-resistant pool. Pools of the transfected BaF3 cells,hereinafter called BaF3/MPL-zalpha11 cells, were assayed for signalingcapability as described below.

B. Testing the Signaling Capability of the BaF3/MPL-zalpha11 Cells Usingan ALAMAR BLUE™ Proliferation Assay

BaF3/MPL-zalpha11 cells were spun down and washed in the complete media,described above, but without mIL-3 (hereinafter referred to as “mIL-3free media”). The cells were spun and washed 3 times to ensure theremoval of the mIL-3 Cells were then counted in a hemacytometer. Cellswere plated in a 96-well format at 5000 cells per well in a volume of100 μl per well using the mIL-3 free media.

Proliferation of the BaF3/MPL-zalpha11 cells was assessed usingthrombopoietin (TPO) diluted with mIL-3 free media to 500 ng/ml, 250ng/ml, 125 ng/ml, 62 ng/ml, 30 ng/ml, 15 ng/ml, 7.5 ng/ml, 3.75 ng/ml,1.8 ng/ml, 0.9 ng/ml, 0.5 ng/ml and 0.25 ng/ml concentrations. 100 μl ofthe diluted TPO was added to the BaF3/MPL-zalpha11 cells. The totalassay volume is 200 μl. Negative controls were run in parallel usingmIL-3 free media only, without the addition of TPO. The assay plateswere incubated at 37° C., 5% CO₂ for 3 days at which time ALAMAR BLUE™(Accumed, Chicago, Ill.) was added at 20 μl/well. Alamar Blue gives afluourometric readout based on number of live cells, and is thus adirect measurement of cell proliferation in comparison to a negativecontrol. Plates were again incubated at 37° C., 5% CO₂ for 24 hours.Plates were read on the FMAX™ plate reader (Molecular Devices Sunnyvale,Calif.) using the SOFTMAX™ Pro program, at wavelengths 544 (Excitation)and 590 (Emmission).

Results confirmed the signaling capability of the intracellular portionof the zalpha11 receptor as the thrombopoietin induced proliferation atapproximately 10 fold over back ground at 62 ng/ml and greater.

Example 6 Construction of Expression Vector Expressing Full-lengthZalpha11

The entire zalpha11 receptor was isolated from a plasmid containingzalpha11 receptor cDNA using PCR with primers ZC19,905 (SEQ ID NO:36)and ZC19,906 (SEQ ID NO:37). The reaction conditions were as follows:95° C. for 1 min; 35 cycles at 95° C. for 1 min, 55° C. for 1 min, 72°C. for 2 min; followed by 72° C. at 10 min; then a 10° C. soak. The PCRproduct was run on a 1% low melting point agarose (Boerhinger Mannheim)and the approximately 1.5 kb zalpha11 cDNA isolated using QIAQUICK™ gelextraction kit (Qiagen) as per manufacturer's instructions.

The purified zalpha11 cDNA was digested with BamHI (Boerhinger Mannheim)and EcoRI (BRL) as per manufacturer's instructions. The entire digestwas run on a 1% low melting point agarose (Boerhinger Mannheim) andpurified the cleaved zalpha11 fragment using QIAQUICK™ gel extractionkit as per manufacturer's instructions. The resultant cleaved zalpha11chimera was inserted into an expression vector as described below.

Recipient expression vector pZP-5N was digested with BamHI (BoerhingerMannheim) and EcoRI (BRL) as per manufacturer's instructions, and gelpurified as described above. This vector fragment was combined with theBamHI and EcoRI cleaved zalpha11 fragment isolated above in a ligationreaction. The ligation was run using T4 Ligase (BRL), at 15° C.overnight. A sample of the ligation was electroporated in to DH10BELECTROMAX™ electrocompetent E. coli cells (25 μF, 200Ω, 2.3V).Transformants were plated on LB+Ampicillin plates and single coloniesscreened by PCR to check for the zalpha11 sequence using ZC19,905 (SEQID NO:36) and ZC19,906 (SEQ ID NO:37) using the PCR conditions asdescribed above.

Confirmation of the MPL-zalpha11 sequence was made by sequence analysesusing the following primers: ZC12,700 (SEQ ID NO:28), ZC5,020 (SEQ IDNO:29), ZC20,114 (SEQ ID NO:38), ZC19,459 (SEQ ID NO:12), ZC19,954 (SEQID NO:39), and ZC20,116 (SEQ ID NO:40). The insert was approximately 1.6kb, and was full-length.

Example 7 Zalpha11 Based Proliferation in BAF3 Assay Using ALAMAR BLUE™

A. Construction of BaF3 Cells Expressing Zalpha11 Receptor

BaF3 cells expressing the full-length zalpha11 receptor were constructedas per Example 5A above, using 30 μg of the zalpha11 expression vector,described in Example 6 above. The BaF3 cells expressing the zalpha11receptor mRNA were designated as BaF3/zalpha11. These cells were used toscreen for a zalpha11 activity as described below in Examples 8 and 12.

Example 8 Screening for Zalpha11 Activity Using BaF3/Zalpha11 CellsUsing an ALAMAR BLUE™ Proliferation Assay

A. Monkey Primary Source Used to Test for Presence of Zalpha11 Activity

Conditioned media from primary monkey spleen cells was used to test forthe presence of activity as described below. Monkey spleen cells wereactivated with 5 ng/ml Phorbol-12-myristate-13-acetate (PMA)(Calbiochem, San Diego, Calif.), and 0.5 μg/ml IONOMYCINT™ ionophore(Calbiochem) for 72 h. The supernatant from the stimulated monkey spleencells was used to assay proliferation of the BaF3/zalpha11 cells asdescribed below.

B. Screening for Zalpha11 Activity Using BaF3/Zalpha11 Cells Using anAlamar Blue™ Proliferation Assay

BaF3/Zalpha11 cells were spun down and washed in mIL-3 free media. Thecells were spun and washed 3 times to ensure the removal of the mIL-3.Cells were then counted in a hemacytometer. Cells were plated in a96-well format at 5000 cells per well in a volume of 100 μl per wellusing the mIL-3 free media.

Proliferation of the BaF3/Zalpha11 cells was assessed using conditionedmedia from activated monkey spleen (see Example 8A, above) was dilutedwith mIL-3 free media to 50%, 25%, 12.5%, 6.25%, 3.125%, 1.5%, 0.75% and0.375% concentrations. 100 μl of the diluted conditioned media was addedto the BaF3/Zalpha11 cells. The total assay volume is 200 μl. The assayplates were incubated at 37° C., 5% CO₂ for 3 days at which time ALAMARBLUE™ dye (Accumed, Chicago, Ill.) was added at 200 μl/well. Plates wereagain incubated at 37° C., 5% CO₂ for 24 hours. Plates were read on theFMAX™ plate reader (Molecular devices) as described above (Example 5).

Results confirmed the proliferative response of the BaF3/Zalpha11 cellsto a factor present in the activate monkey spleen conditioned media. Theresponse, as measured, was approximately 4-fold over background at the50% concentration. The BaF3 wild type cells did not proliferate inresponse to this factor, showing that this factor is specific for theZalpha11 receptor.

C. Human Primary Source Used to Isolate Zalpha11 Activity

100 ml blood draws were taken from each of six donors. The blood wasdrawn using 10×10 ml vacutainer tubes containing heparin. Blood waspooled from six donors (600 ml), diluted 1:1 in PBS, and separated usinga FICOLL-PAQUE® PLUS solution (Pharmacia Biotech, Uppsala, Sweden). Theisolated primary human cell yield after separation on the ficollgradient was 1.2×10⁹ cells.

Cells were suspended in 9.6 ml MACS buffer (PBS, 0.5% EDTA, 2 mM EDTA).1.6 ml of cell suspension was removed and 0.4 ml CD3 microbeads(Miltenyi Biotec, Auburn, Calif.) added. The mixture was incubated for15 min. at 4° C. These cells labeled with CD3 beads were washed with 30ml MACS buffer, and then resuspended in 2 ml MACS buffer.

A VS+ column (Miltenyi) was prepared according to the manufacturer'sinstructions. The VS+ column was then placed in a VARIOMACS™ magneticfield (Miltenyi). The column was equilibrated with 5 ml MACS buffer. Theisolated primary human cells were then applied to the column. The CD3negative cells were allowed to pass through. The column was rinsed with9 ml (3×3 ml) MACS buffer. The column was then removed from the magnetand placed over a 15 ml falcon tube. CD3+ cells were eluted by adding 5ml MACS buffer to the column and bound cells flushed out using theplunger provided by the manufacturer. The incubation of the cells withthe CD3 magnetic beads, washes, and VS+ column steps (incubation throughelution) above were repeated five more times. The resulting CD3+fractions from the six column separations were pooled. The yield of CD3+selected human T-cells were 3×10⁸ total cells.

A sample of the pooled CD3+ selected human T-cells was removed forstaining and sorting on a fluorescent antibody cell sorter (FACS) toassess their purity. The CD3+ selected human T-cells were 91% CD3+cells.

The CD3+ selected human T-cells were activated by incubating in RPMI+5%FBS+PMA 10 ng/ml and Ionomycin 0.5 μg/ml (Calbiochem) for 13 hours 37°C. The supernatant from these activated CD3+ selected human T-cells wastested for zalpha11 activity as described below.

D. Testing Supernatant from Activated CD3+ Selected Human T-cells forZalpha11 Activity Using BaF3/Zalpha11 Cells and an ALAMAR BLUE™Proliferation Assay

BaF3/Zalpha11 cells were spun down and washed in mIL-3 free media. Thecells were spun and washed 3 times to ensure the removal of the mIL-3.Cells were then counted in a hemacytometer. Cells were plated in a96-well format at 5000 cells per well in a volume of 100 μl per wellusing the mIL-3 free media.

Proliferation of the BaF3/Zalpha11 cells was assessed using conditionedmedia from activated CD3+ selected human T-cells (see Example 8C, above)diluted with mIL-3 free media to 50%, 25%, 12.5%, 6.25%, 3.125%, 1.5%,0.75% and 0.375% concentrations. 100 μl of the diluted conditioned mediawas added to the BaF3/Zalpha11 cells. The total assay volume is 200 μl.The assay plates were incubated and assayed as described in Example 8Babove.

Results confirmed the proliferative response of the BaF3/Zalpha11 cellsto a factor present in the activated CD3+ selected human T-cellconditioned media. The response, as measured, was approximately 10-foldover background at the 50% concentration. The BaF3 wild type cells didnot proliferate in response to this factor, showing that this factor isspecific for the Zalpha11 receptor.

Example 9 Construction of Mammalian Expression Vectors that ExpressZalpha11 Soluble Receptors: Zalpha11CEE, Zalpha11CFLG, Zalpha11CHIS andZalph11-Fc4

A. Construction of Zalpha11 Mammalian Expression Vector containingZalph11CEE, Zalph11CFLG and Zalph11CHIS

An expression vector was prepared for the expression of the soluble,extracellular domain of the zalpha11 polypeptide, pC4zalph11CEE, whereinthe construct is designed to express a zalpha11 polypeptide comprised ofthe predicted initiating methionine and truncated adjacent to thepredicted transmembrane domain, and with a C-terminal Glu-Glu tag (SEQID NO:41).

A 700 bp PCR generated zalpha11 DNA fragment was created using ZC19,931(SEQ ID NO:42) and ZC19,932 (SEQ ID NO:43) as PCR primers to add Asp718and BamHI restriction sites. A plasmid containing the zalpha11 receptorcDNA was used as a template. PCR amplification of the zalpha11 fragmentwas performed as follows: Twenty five cycles at 94C for 0.5 minutes;five cycles at 94° C. for 10 seconds, 50° C. for 30 seconds, 68° C. for45 seconds, followed by a 4° C. hold. The reaction was purified bychloroform/phenol extraction and isopropanol precipitation, and digestedwith Asp718 and BamHI (Gibco BRL) following manufacturer's protocol. Aband of the predicted size, 700 bp, was visualized by 1% agarose gelelectrophoresis, excised and the DNA was purified using a QIAEXII™purification system (Qiagen) according the manufacturer's instructions.

The excised DNA was subcloned into plasmid pC4EE which had been cut withBamHI and Asp718. The pC4zalph11CEE expression vector uses the nativezalpha11 signal peptide and attaches the Glu-Glu tag (SEQ ID NO:41) tothe C-terminus of the zalpha11 polypeptide-encoding polynucleotidesequence. Plasmid pC4EE, is a mammalian expression vector containing anexpression cassette having the mouse metallothionein-1 promoter,multiple restriction sites for insertion of coding sequences, a stopcodon and a human growth hormone terminator. The plasmid also has an E.coli origin of replication, a mammalian selectable marker expressionunit having an SV40 promoter, enhancer and origin of replication, a DHFRgene and the SV40 terminator.

About 30 ng of the restriction digested zalpha11 insert and about 12 ngof the digested vector were ligated overnight at 16° C. One microliterof each ligation reaction was independently electroporated into DH10Bcompetent cells (GIBCO BRL, Gaithersburg, Md.) according tomanufacturer's direction and plated onto LB plates containing 50 mg/mlampicillin, and incubated overnight. Colonies were screened byrestriction analysis of DNA prepared from 2 ml liquid cultures ofindividual colonies. The insert sequence of positive clones was verifiedby sequence analysis. A large scale plasmid preparation was done using aQIAGEN® Maxi prep kit (Qiagen) according to manufacturer's instructions.

The same process was used to prepare the zalpha11 soluble receptors witha C-terminal his tag, composed of 6 His residues in a row; and aC-terminal flag (SEQ ID NO:49) tag, zalpha11CFLAG. To construct theseconstructs, the aforementioned vector has either the HIS or the FLAG®tag in place of the glu-glu tag (SEQ ID NO:41).

B. Mammalian Expression Construction of Soluble Zalpha11 receptorZalpha11-Fc4

An expression plasmid containing all or part of a polynucleotideencoding zalpha11 was constructed via homologous recombination. Afragment of zalpha11 cDNA was isolated using PCR that includes thepolynucleotide sequence from extracellular domain of the zalpha11receptor. The two primers used in the production of the zalpha11fragment were: (1) The primers for PCR each include from 5′ to 3′ end:40 bp of the vector flanking sequence (5′ of the insert) and 17 bpcorresponding to the 5′ end of the zalpha11 extracellular domain (SEQ IDNO:44); and (2) 40 bp of the 5′ end of the Fc4 polynucleotide sequence(SEQ ID NO:45) and 17 bp corresponding to the 3′ end of the zalpha11extracellular domain (SEQ ID NO:46). The fragment of Fc-4 for fusionwith the zalpha11 was generated by PCR in a similar fashion. The twoprimers used in the production of the Fc4 fragment were: (1) a 5′ primerconsisting of 40 bp of sequence from the 3′ end of zalpha11extracellular domain and 17 bp of the 5′ end of Fc4 (SEQ ID NO:47); and(2) a 3′ primer consisting of 40 bp of vector sequence (3′ of theinsert) and 17 bp of the 3′ end of Fc4 (SEQ ID NO:48).

PCR amplification of the each of the reactions described above wasperformed as follows: one cycle at 94° C. for 2 minutes; twenty-fivecycles at 94° C. for 30 seconds, 60° C. for 30 seconds, 72° C. for 1minute; one cycle at 72° C. for 5 minutes; followed by a 4° C. hold. Tenμl of the 100 μl PCR reaction was run on a 0.8% LMP agarose gel(Seaplaque GTG) with 1×TBE buffer for analysis. The remaining 90 μl ofPCR reaction is precipitated with the addition of 5 μl 1 M NaCl and 250μl of absolute ethanol. The expression vector used was derived from theplasmid pCZR199 (deposited at the American Type Culture Collection,10801 University Boulevard, Manassas, Va. 20110-2209, and is designatedNo. 98668), and was cut with SmaI (BRL). The expression vector wasderived from the plasmid pCZR199, and is a mammalian expression vectorcontaining an expression cassette having the CMV immediate earlypromoter, a consensus intron from the variable region of mouseimmunoglobulin heavy chain locus, multiple restriction sites forinsertion of coding sequences, a stop codon and a human growth hormoneterminator. The expression vector also has an E. coli origin ofreplication, a mammalian selectable marker expression unit having anSV40 promoter, enhancer and origin of replication, a DHFR gene and theSV40 terminator. The expression vector used was constructed from pCZR199by the replacement of the metallothionein promoter with the CMVimmediate early promoter.

One hundred microliters of competent yeast cells (S. cerevisiae) werecombined with 10 μl containing approximately 1 μg each of the zalpha11and Fc4 inserts, and 100 ng of SmaI (BRL) digested expression vector andtransferred to a 0.2 cm electroporation cuvette. The yeast/DNA mixtureswere electropulsed at 0.75 kV (5 kV/cm), “infinite” ohms, 25 μF. To eachcuvette is added 600 μl of 1.2 M sorbitol and the yeast was plated intwo 300 μl aliquots onto two URA-D plates and incubated at 30° C.

After about 48 hours, the Ura+yeast transformants from a single platewere resuspended in 1 ml H₂O and spun briefly to pellet the yeast cells.The cell pellet was resuspended in 1 ml of lysis buffer (2% TritonX-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Five hundredmicroliters of the lysis mixture was added to an Eppendorf tubecontaining 300 μl acid washed glass beads and 200 μl phenol-chloroform,vortexed for 1 minute intervals two or three times, followed by a 5minute spin in a Eppendorf centrifuge at maximum speed. Three hundredmicroliters of the aqueous phase was transferred to a fresh tube, andthe DNA precipitated with 600 μl ethanol (EtOH), followed bycentrifugation for 10 minutes at 4° C. The DNA pellet was resuspended in100 μl H₂O.

Transformation of electrocompetent E. coli cells (DH10B, GibcoBRL) isdone with 0.5-2 ml yeast DNA prep and 40 ul of DH10B cells. The cellswere electropulsed at 2.0 kV, 25 mF and 400 ohms. Followingelectroporation, 1 ml SOC (2% Bactoë Tryptone (Difco, Detroit, Mich.),0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mMMgSO4, 20 mM glucose) was plated in 250 μl aliquots on four LB AMPplates (LB broth (Lennox), 1.8% Bacto Agar (Difco), 100 mg/LAmpicillin).

Individual clones harboring the correct expression construct forzalpha11-Fc4 were identified by restriction digest to verify thepresence of the zalpha11-Fc4 insert and to confirm that the various DNAsequences have been joined correctly to one another. The insert ofpositive clones were subjected to sequence analysis. Larger scaleplasmid DNA is isolated using the Qiagen Maxi kit (Qiagen) according tomanufacturer's instructions.

Example 10 Transfection And Expression of Zalpha11 Soluble ReceptorPolypeptides

BHK 570 cells (ATCC No. CRL-10314), passage 27, were plated at 1.2×10⁶cells/well (6-well plate) in 800 μl of serum free (SF) DMEM media (DMEM,Gibco/BRL High Glucose) (Gibco BRL, Gaithersburg, Md.). The cells weretransfected with expression plasmids containing zalpha11 CEE/CFLG/CHISdescribed above (see, Example 9), using LIPOFECTIN™ reagent (Gibco BRL),in serum free (SF) DMEM. Three micrograms of zalpha11 CEE/CFLG/CHIS eachwere separately diluted into 1.5 ml tubes to a total final volume of 100μl SF DMEM. In separate tubes, 15 μl of LIPOFECTIN™ reagent (Gibco BRL)was mixed with 100 μl of SF DMEM. The LIPOFECTIN™ reagent mix wasincubated at room temperature for 30-45 minutes then the DNA mix wasadded and allowed to incubate approximately 10-15 minutes at roomtemperature.

The entire DNA:LIPOFECTIN™ reagent mixture was added to the plated cellsand distributed evenly over them. The cells were incubated at 37° C. forapproximately five hours, then transferred to separate 150 mm MAXIplates in a final volume of 30 ml DMEM/5% fetal bovine serum (FBS)(Hyclone, Logan, Utah). The plates were incubated at 37° C., 5% CO₂,overnight and the DNA:LIPOFECTIN™ reagent mixture was replaced withselection media (5% FBS/DMEM with 1 μM methotrexate (MTX)) the next day.

Approximately 10-12 days post-transfection, the plates were washed with10 ml SF DMEM. The wash media was aspirated and replaced with 7.25 mlserum-free DMEM. Sterile Teflon meshes (Spectrum Medical Industries, LosAngeles, Calif.) pre-soaked in SF DMEM were then placed over the clonalcell colonies. A sterile nitrocellulose filter pre-soaked in SF DMEM wasthen placed over the mesh. Orientation marks on the nitrocellulose weretransferred to the culture dish. The plates were then incubated for 5-6hours in a 37° C., 5% CO₂ incubator.

Following incubation, the filters/meshes were removed, and the mediaaspirated and replaced with 5% FBS/DMEM with 1 μM MTX. The filters werethen blocked in 10% nonfat dry milk/Western A buffer (Western A: 50 mMTris pH 7.4, 5 mM EDTA, 0.05% NP-40, 150 mM NaCl and 0.25% gelatin) for15 minutes at room temperature on a rotating shaker. The filters werethen incubated with an anti-Glu-Glu, anti-FLAG®, or anti-HISantibody-HRP conjugates, respectively, in 2.5% nonfat dry milk/Western Abuffer for one hour at room temperature on a rotating shaker. Thefilters were then washed three times at room temperature with Western Afor 5-10 minutes per wash. The filters were developed with ultra ECLreagent (Amersham Corp., Arlington Heights, Ill.) according themanufacturer's directions and visualized on the Lumi-Imager (RocheCorp.)

Positive expressing clonal colonies were mechanically picked to 12-wellplates in one ml of 5% FCS/DMEM with 5 μM MTX, then grown to confluence.Conditioned media samples were then tested for expression levels viaSDS-PAGE and Western analysis. The three highest expressing clones foreach construct were picked; two out of three were frozen down as back upand one was expanded for mycoplasma testing and large-scale factoryseeding.

B. Mammalian Expression of Soluble Zalpha11 Receptor Zalpha11-Fc4

BHK 570 cells (ATCC NO: CRL-10314) were plated in 10 cm tissue culturedishes and allowed to grow to approximately 50 to 70% confluencyovernight at 37° C., 5% CO₂, in DMEM/FBS media (DMEM, Gibco/BRL HighGlucose, (Gibco BRL, Gaithersburg, Md.), 5% fetal bovine serum (Hyclone,Logan, Utah), 1 mM L-glutamine (JRH Biosciences, Lenexa, Kans.), 1 mMsodium pyruvate (Gibco BRL)). The cells were then transfected with theplasmid containing zalpha11-Fc4 (see, Example 9), using LIPOFECTAMINE™(Gibco BRL), in serum free (SF) media formulation (DMEM, 10 mg/mltransferrin, 5 mg/ml insulin, 2 mg/ml fetuin, 1% L-glutamine and 1%sodium pyruvate). The plasmid containing zalpha11-Fc4 was diluted into15 ml tubes to a total final volume of 640 ml with SF media. 35 ml ofLIPOFECTAMINE™ (Gibco BRL) was mixed with 605 ml of SF medium. TheLIPOFECTAMINE™ mix was added to the DNA mix and allowed to incubateapproximately 30 minutes at room temperature. Five milliliters of SFmedia was added to the DNA:LIPOFECTAMINE™ mixture. The cells were rinsedonce with 5 ml of SF media, aspirated, and the DNA:LIPOFECTAMINE™mixture is added. The cells were incubated at 37° C. for five hours,then 6.4 ml of DMEM/10% FBS, 1% PSN media was added to each plate. Theplates were incubated at 37° C. overnight and the DNA:LIPOFECTAMINE™mixture was replaced with fresh 5% FBS/DMEM media the next day. On day 2post-transfection, the cells were split into the selection media(DMEM/FBS media from above with the addition of 1 mM methotrexate (SigmaChemical Co., St. Louis, Mo.)) in 150 mm plates at 1:10, 1:20 and 1:50.The media on the cells was replaced with fresh selection media at day 5post-transfection. Approximately 10 days post-transfection, two 150 mmculture dishes of methotrexate resistant colonies from each transfectionwere trypsinized and the cells are pooled and plated into a T-162 flaskand transferred to large scale culture.

Example 11 Purification of Zalpha11 Soluble Rreceptors from BHK 570Cells

A. Purification of Zalpha11CEE Polypeptide from BHK 570

Unless otherwise noted, all operations were carried out at 4° C. Thefollowing procedure was used for purifying zalpha11 polypeptidecontaining C-terminal GluGlu (EE) tags. Thirty liters of cell factoryconditioned media was concentrated to 1.6 liters with an Amicon S10Y3spiral cartridge on a ProFlux A30. A Protease inhibitor solution wasadded to the concentrated 1.6 liters of cell factory conditioned mediafrom transfected BHK 570 cells (see, Example 10) to final concentrationsof 2.5 mM ethylenediaminetetraacetic acid (EDTA, Sigma Chemical Co. St.Louis, Mo.), 0.003 mM leupeptin (Boehringer-Mannheim, Indianapolis,Ind.), 0.001 mM pepstatin (Boehringer-Mannheim) and 0.4 mM Pefabloc(Boehringer-Mannheim). Samples were removed for analysis and the bulkvolume was frozen at −80° C. until the purification was started. Totaltarget protein concentrations of the concentrated cell factoryconditioned media was determined via SDS-PAGE and Western blot analysiswith the anti-EE HRP conjugated antibody.

A 100 ml column of anti-EE G-Sepharose (prepared as described below) waspoured in a Waters AP-5, 5 cm×10 cm glass column. The column was flowpacked and equilibrated on a BioCad Sprint (PerSeptive BioSystems,Framingham, Mass.) with phosphate buffered saline (PBS) pH 7.4. Theconcentrated cell factory conditioned media was thawed, 0.2 micronsterile filtered, pH adjusted to 7.4, then loaded on the columnovernight with 1 ml/minute flow rate. The column was washed with 10column volumes (CVs) of phosphate buffered saline (PBS, pH 7.4), thenplug eluted with 200 ml of PBS (pH 6.0) containing 0.5 mg/ml EE peptide(Anaspec, San Jose, Calif.) at 5 ml/minute. The EE peptide used has thesequence EYMPME (SEQ ID NO:41). The column was washed for 10 CVs withPBS, then eluted with 5 CVs of 0.2M glycine, pH 3.0. The pH of theglycine-eluted column was adjusted to 7.0 with 2 CVs of 5×PBS, thenequilibrated in PBS (pH 7.4). Five ml fractions were collected over theentire elution chromatography and absorbance at 280 and 215 nM weremonitored; the pass through and wash pools were also saved and analyzed.The EE-polypeptide elution peak fractions were analyzed for the targetprotein via SDS-PAGE Silver staining and Western Blotting with theanti-EE HRP conjugated antibody. The polypeptide elution fractions ofinterest were pooled and concentrated from 60 ml to 5.0 ml using a10,000 Dalton molecular weight cutoff membrane spin concentrator(Millipore, Bedford, Mass.) according to the manufacturer'sinstructions.

To separate zalpha11CEE from other co-purifying proteins, theconcentrated polypeptide elution pooled fractions were subjected to aPOROS HQ-50 (strong anion exchange resin from PerSeptive BioSystems,Framingham, Mass.) at pH 8.0. A 1.0×6.0 cm column was poured and flowpacked on a BioCad Sprint. The column was counter ion charged thenequibrated in 20 mM TRIS pH 8.0 (Tris (Hydroxymethyl Aminomethane)). Thesample was diluted 1:13 (to reduce the ionic strength of PBS) thenloaded on the Poros HQ column at 5 ml/minute. The column was washed for10 CVs with 20 mM Tris pH 8.0 then eluted with a 40 CV gradient of 20 mMTris/1 M sodium chloride (NaCl) at 10 ml/minute. 1.5 ml fractions werecollected over the entire chromatography and absorbance at 280 and 215nM were monitored. The elution peak fractions were analyzed via SDS-PAGESilver staining Fractions of interest were pooled and concentrated to1.5-2 ml using a 10,000 Dalton molecular weight cutoff membrane spinconcentrator (Millipore, Bedford, Mass.) according to the manufacturer'sinstructions.

To separate zalpha11CEE polypeptide from free EE peptide and anycontaminating co-purifying proteins, the pooled concentrated fractionswere subjected to chromatography on a 1.5×90 cm Sephadex 5200(Pharmacia, Piscataway, N.J.) column equilibrated and loaded in PBS at aflow rate of 1.0 ml/min using a BioCad Sprint. 1.5 ml fractions werecollected across the entire chromatography and the absorbance at 280 and215 nM were monitored. The peak fractions were characterized viaSDS-PAGE Silver staining, and only the most pure fractions were pooled.This material represented purified zalpha11CEE polypeptide.

This purified material was finally subjected to a 4 ml ActiClean Etox(Sterogene) column to remove any remaining endotoxins. The sample waspassed over the PBS equilibrated gravity column four times then thecolumn was washed with a single 3 ml volume of PBS, which was pooledwith the “cleaned” sample. The material was then 0.2 micron sterilefiltered and stored at −80° C. until it was aliquoted.

On Western blotted, Coomassie Blue and Silver stained SDS-PAGE gels, thezalpha11CEE polypeptide was one major band of an apparent molecularweight of 50,000 Daltons. The mobility of this band was the same onreducing and non-reducing gels.

The protein concentration of the purified material was performed by BCAanalysis (Pierce, Rockford, Ill.) and the protein was aliquoted, andstored at −80° C. according to our standard procedures. On IEF(isoelectric focusing) gels the protein runs with a PI of less than 4.5.The concentration of zalpha11CEE polypeptide was 1.0 mg/ml.

Purified zalpha11CEE polypeptide was prepared for injection into rabbitsand sent to R & R Research and Development (Stanwood, Wash.) forantibody production. Rabbits were injected to produceanti-huzalpha11-CEE-BHK serum (Example 15, below).

To prepare anti-EE Sepharose, a 100 ml bed volume of protein G-Sepharose(Pharmacia, Piscataway, N.J.) was washed 3 times with 100 ml of PBScontaining 0.02% sodium azide using a 500 ml Nalgene 0.45 micron filterunit. The gel was washed with 6.0 volumes of 200 mM triethanolamine, pH8.2 (TEA, Sigma, St. Louis, Mo.), and an equal volume of EE antibodysolution containing 900 mg of antibody was added. After an overnightincubation at 4° C., unbound antibody was removed by washing the resinwith 5 volumes of 200 mM TEA as described above. The resin wasresuspended in 2 volumes of TEA, transferred to a suitable container,and dimethylpimilimidate-2HCl (Pierce, Rockford, Ill.) dissolved in TEA,was added to a final concentration of 36 mg/ml of protein G-Sepharosegel. The gel was rocked at room temperature for 45 min and the liquidwas removed using the filter unit as described above. Nonspecific siteson the gel were then blocked by incubating for 10 min. at roomtemperature with 5 volumes of 20 mM ethanolamine in 200 mM TEA. The gelwas then washed with 5 volumes of PBS containing 0.02% sodium azide andstored in this solution at 4° C.

B. Purification of Zalpha11CFLAG Polypeptide from BHK 570

Unless otherwise noted, all operations were carried out at 4° C. Thefollowing procedure was used for purifying zalpha11 polypeptidecontaining C-terminal FLAG® (FLG) (Sigma-Aldrich Co.) tags. Thirtyliters of cell factory conditioned media was concentrated to 1.7 literswith an Amicon S10Y3 spiral cartridge on a ProFlux A30. A Proteaseinhibitor solution was added to the 1.7 liters of concentrated cellfactory conditioned media from transfected BHK 570 cells (see, Example10) to final concentrations of 2.5 mM ethylenediaminetetraacetic acid(EDTA, Sigma Chemical Co. St. Louis, Mo.), 0.003 mM leupeptin(Boehringer-Mannheim, Indianapolis, Ind.), 0.001 mM pepstatin(Boehringer-Mannheim) and 0.4 mM Pefabloc (Boehringer-Mannheim). Sampleswere removed for analysis and the bulk volume was frozen at −80° C.until the purification was started. Total target protein concentrationsof the cell factory conditioned media was determined via SDS-PAGE andWestern blot analysis with the anti-FLAG® (Kodak) HRP conjugatedantibody. A 125 ml column of anti-FLAG® M2-Agarose affinity gel(Sigma-Aldrich Co.) was poured in a Waters AP-5, 5 cm×10 cm glasscolumn. The column was flow packed and equilibrated on a BioCad Sprint(PerSeptive BioSystems, Framingham, Mass.) with phosphate bufferedsaline (PBS) pH 7.4. The concentrated cell factory conditioned media wasthawed, 0.2 micron sterile filtered, pH adjusted to 7.4, then loaded onthe column overnight with 1 ml/minute flow rate. The column was washedwith 10 column volumes (CVs) of phosphate buffered saline (PBS, pH 7.4),then plug eluted with 250 ml of PBS (pH 6.0) containing 0.5 mg/ml FLAG®(Sigma-Aldrich Co.) peptide at 5 ml/minute. The FLAG® peptide used hasthe sequence DYKDDDDK (SEQ ID NO:49). The column was washed for 10 CVswith PBS, then eluted with 5 CVs of 0.2M glycine, pH 3.0. The pH of theglycine-eluted column was adjusted to 7.0 with 2 CVs of 5×PBS, thenequilibrated in PBS (pH 7.4). Five ml fractions were collected over theentire elution chromatography and absorbence at 280 and 215 nM weremonitored; the pass through and wash pools were also saved and analyzed.The FLAG®-polypeptide elution peak fractions were analyzed for thetarget protein via SDS-PAGE Silver staining and Western Blotting withthe anti-FLAG HRP conjugated antibody. The polypeptide elution fractionsof interest were pooled and concentrated from 80 ml to 12 ml using a10,000 Dalton molecular weight cutoff membrane spin concentrator(Millipore, Bedford, Mass.) according to the manufacturer'sinstructions.

To separate zalpha11CFLG from other co-purifying proteins, thepolypeptide elution pooled fractions were subjected to a POROS HQ-50(strong anion exchange resin from PerSeptive BioSystems, Framingham,Mass.) at pH 8.0. A 1.0×6.0 cm column was poured and flow packed on aBioCad Sprint. The column was counter ion charged then equilibrated in20 mM TRIS pH 8.0 (Tris (Hydroxymethyl Aminomethane)). The sample wasdiluted 1:13 (to reduce the ionic strength of PBS) then loaded on thePoros HQ-50 column at 5 ml/minute. The column was washed for 10 columnvolumes (CVs) with 20 mM Tris pH 8.0 then eluted with a 40 CV gradientof 20 mM Tris/1 M sodium chloride (NaCl) at 10 ml/minute. 1.5 mlfractions were collected over the entire chromatography and absorbanceat 280 and 215 nM were monitored. The elution peak fractions wereanalyzed via SDS-PAGE Silver staining Fractions of interest were pooledand concentrated to 1.5-2 ml using a 10,000 Dalton molecular weightcutoff membrane spin concentrator (Millipore, Bedford, Mass.) accordingto the manufacturer's instructions.

To separate zalpha11CFLG polypeptide from free FLAG® peptide and anycontaminating co-purifying proteins, the pooled concentrated fractionswere subjected to chromatography on a 1.5×90 cm Sephacryl 5200(Pharmacia, Piscataway, N.J.) column equilibrated and loaded in PBS at aflow rate of 1.0 ml/min using a BioCad Sprint. 1.5 ml fractions werecollected across the entire chromatography and the absorbance at 280 and215 nM were monitored. The peak fractions were characterized viaSDS-PAGE Silver staining, and only the most pure fractions were pooled.This material represented purified zalpha11CFLG polypeptide.

This purified material was finally subjected to a 4 ml ActiClean Etox(Sterogene) column to remove any remaining endotoxins. The sample waspassed over the PBS equilibrated gravity column four times then thecolumn was washed with a single 3 ml volume of PBS, which was pooledwith the “cleaned” sample. The material was then 0.2 micron sterilefiltered and stored at −80° C. until it was aliquoted.

On Western blotted, Coomassie Blue and Silver stained SDS-PAGE gels, thezalpha11CFLG polypeptide was one major band of an apparent molecularweight of 50,000 Daltons. The mobility of this band was the same onreducing and non-reducing gels.

The protein concentration of the purified material was performed by BCAanalysis (Pierce, Rockford, Ill.) and the protein was aliquoted, andstored at −80° C. according to our standard procedures. On IEF(isoelectric focusing) gels the protein runs with a PI of less than 4.5.The concentration of zalpha11CFLG polypeptide was 1.2 mg/ml.

C. Purification of Zalpha11-Fc4 Polypeptide from Transfected BHK 570Cells

Unless otherwise noted, all operations were carried out at 4° C. Thefollowing procedure was used for purifying zalpha11 polypeptidecontaining C-terminal fusion to human IgG/Fc (zalpha11-Fc4; Examples 8and 9). 12,000 ml of conditioned media from BHK 570 cells transfectedwith zalpha11-Fc4 (Example 10) was filtered through a 0.2 mm sterilizingfilter and then supplemented with a solution of protease inhibitors, tofinal concentrations of, 0.001 mM leupeptin (Boerhinger-Mannheim,Indianapolis, Ind.), 0.001 mM pepstatin (Boerhinger-Mannheim) and 0.4 mMPefabloc (Boerhinger-Mannheim). A protein G sepharose (6 ml bed volume,Pharmacia Biotech) was packed and washed with 500 ml PBS (Gibco/BRL) Thesupplemented conditioned media was passed over the column with a flowrate of 10 ml/minute, followed by washing with 1000 ml PBS (BRL/Gibco).zalpha11-Fc4 was eluted from the column with 0.1 M Glycine pH 3.5 and 2ml fractions were collected directly into 0.2 ml 2M Tris pH 8.0, toadjust the final pH to 7.0 in the fractions.

The eluted fractions were characterized by SDS-PAGE and western blottingwith anti-human Fc (Amersham) antibodies. Western blot analysis ofreducing SDS-PAGE gels reveal an immunoreactive protein of 80,000 KDa infractions 2-10. Silver stained SDS-PAGE gels also revealed an 80,000 KDazalpa11:Fc polypeptide in fractions 2-10. Fractions 2-10 were pooled.

The protein concentration of the pooled fractions was performed by BCAanalysis (Pierce, Rockford, Ill.) and the material was aliquoted, andstored at −80° C. according to our standard procedures. Theconcentration of the pooled fractions was 0.26 mg/ml.

Example 12 Assay Using Zalpha11 Soluble Receptor Zalpha11CEE,Zalpha11CFLG and Zalpha11-Fc4 (Mutant) Soluble Receptors in CompetitiveInhibition Assay

BaF3/Zalpha11 cells were spun down and washed in mIL-3 free media. Thecells were spun and washed 3 times to ensure the removal of the mIL-3.Cells were then counted in a hemacytometer. Cells were plated in a96-well format at 5000 cells per well in a volume of 100 ml per wellusing the mIL-3 free media.

Both media from the monkey spleen cell activation and the CD3+ selectedcells, described in Example 8 above, were added in separate experimentsat 50%, 25%, 12.5%, 6.25%, 3.125%, 1.5%, 0.75% and 0.375%concentrations, with or without zalpha11 soluble receptors (CEE, C-flag,and Fc4 constructs; See, Example 10 and 11) at 10 μg/ml. The total assayvolume was 200

The assay plates were incubated 37° C., 5% CO₂ for 3 days at which timeAlamar Blue (Accumed) was added at 20 μl/well. Plates were againincubated at 37° C., 5% CO₂ for 24 hours. Plates were read on the Fmax™plate reader (Molecular Devices) as described above (Example 5). Resultsdemonstrated complete inhibition of cell growth from each of thedifferent zalpha11 soluble receptor constructs at 10 μg/ml, confirmingthat the factor in each sample was specific for the zalpha11 receptor.

Titration curves, diluting out the soluble receptors, were also runusing the above stated assay. Both the zalpha11CEE and zalpha11CFLGsoluble zalpha11 receptors were able to completely inhibit growth as lowas 20 ng/ml. The mutant zalpha11-Fc4 soluble zalpha11 receptor was onlyas effective at 1.5 μg/ml.

Example 13 Expression of Human Zalpha11 in E. coli

A. Construction of Expression Vector pCZR225 that ExpressesHuzalpha11/MBP-6H Fusion Polypeptide

An expression plasmid containing a polynucleotide encoding a humanzalpha11 soluble receptor fused C-terminally to maltose binding protein(MBP) was constructed via homologous recombination. The polynucleotidesequence for the MBP-zalpha11 soluble receptor fusion polypeptide isshown in SEQ ID NO:50, with the corresponding protein sequence shown inSEQ ID NO:51. The fusion polypeptide, designated huzalpha11/MBP-6H, inExample 14, contains an MBP portion (amino acid 1 (Met) to amino acid388 (Ser) of SEQ ID NO:51) fused to the human zalpha11 soluble receptor(amino acid 389 (Cys) to amino acid 606 (His) of SEQ ID NO:51). Afragment of human zalpha11 cDNA (SEQ ID NO:52) was isolated using PCR.Two primers were used in the production of the human zalpha11 fragmentin a PCR reaction: (1) Primer ZC20,187 (SEQ ID NO:53), containing 40 bpof the vector flanking sequence and 25 bp corresponding to the aminoterminus of the human zalpha11, and (2) primer ZC20,185 (SEQ ID NO:54),containing 40 bp of the 3′ end corresponding to the flanking vectorsequence and 25 bp corresponding to the carboxyl terminus of the humanzalpha11. The PCR Reaction conditions were as follows: 25 cycles of 94°C. for 30 seconds, 50° C. for 30 seconds, and 72° C. for 1 minute;followed by 4° C. soak, run in duplicate. Two μl of the 100 μl PCRreaction was run on a 1.0% agarose gel with 1×TBE buffer for analysis,and the expected approximately 660 bp fragment was seen. The remaining90 μl of PCR reaction was combined with the second PCR tube precipitatedwith 400 μl of absolute ethanol. The precipitated DNA used forrecombining into the SmaI cut recipient vector pTAP98 to produce theconstruct encoding the MBP-zalpha11 fusion, as described below.

Plasmid pTAP98 was derived from the plasmids pRS316 and pMAL-c2. Theplasmid pRS316 is a Saccharomyces cerevisiae shuttle vector (Hieter P.and Sikorski, R., Genetics 122:19-27, 1989). pMAL-C2 (NEB) is an E. coliexpression plasmid. It carries the tac promoter driving MalE (geneencoding MBP) followed by a His tag, a thrombin cleavage site, a cloningsite, and the rrnB terminator. The vector pTAP98 was constructed usingyeast homologous recombination. 100 ng of EcoR1 cut pMAL-c2 wasrecombined with 1 μg Pvu1 cut pRS316, 1 μg linker, and 1 μg Sca1/EcoR1cut pRS316. The linker consisted of oligos ZC19,372 (SEQ ID NO:55) (100μmol): ZC19,351 (SEQ ID NO:56) (1 μmol): ZC19,352 (SEQ ID NO:57) (1μmol), and ZC19,371 (SEQ ID NO:58) (100 μmol) combined in a PCRreaction. PCR reaction conditions were as follows: 10 cycles of 94° C.for 30 seconds, 50° C. for 30 seconds, and 72° C. for 30 seconds;followed by 4° C. soak. PCR products were concentrated via 100% ethanolprecipitation.

One hundred microliters of competent yeast cells (S. cerevisiae) werecombined with 10 μl of a mixture containing approximately 1 μg of thehuman zalpha11 receptor PCR product above, and 100 ng of SmaI digestedpTAP98 vector, and transferred to a 0.2 cm electroporation cuvette. Theyeast/DNA mixture was electropulsed at 0.75 kV (5 kV/cm), infinite ohms,25 μF. To each cuvette was added 600 μl of 1.2 M sorbitol and the yeastwas then plated in two 300 μl aliquots onto two-URA D plates andincubated at 30° C.

After about 48 hours, the Ura+ yeast transformants from a single platewere resuspended in 1 ml H₂O and spun briefly to pellet the yeast cells.The cell pellet was resuspended in 1 ml of lysis buffer (2% TritonX-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Five hundredmicroliters of the lysis mixture was added to an Eppendorf tubecontaining 300 μl acid washed glass beads and 200 μl phenol-chloroform,vortexed for 1 minute intervals two or three times, followed by a 5minute spin in a Eppendorf centrifuge at maximum speed. Three hundredmicroliters of the aqueous phase was transferred to a fresh tube, andthe DNA precipitated with 600 μl ethanol (EtOH), followed bycentrifugation for 10 minutes at 4° C. The DNA pellet was resuspended in100 μl H₂O.

Transformation of electrocompetent E. coli cells (MC1061, Casadaban et.al. J. Mol. Biol. 138, 179-207) was done with 1 μl yeast DNA prep and 40μl of MC1061 cells. The cells were electropulsed at 2.0 kV, 25 μF and400 ohms. Following electroporation, 0.6 ml SOC (2% Bacto™ Tryptone(Difco, Detroit, Mich.), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mMKCl, 10 mM MgCl₂, 10 mM MgSO4, 20 mM glucose) was plated in one aliquoton MM/CA +AMP 100 mg/L plates (Pryor and Leiting, Protein Expression andPurification 10:309-319, 1997).

Cells harboring the correct expression construct for human zalpha11receptor were identified by expression. Cells were grown in MM/CA with100 μg/ml Ampicillin for two hours, shaking, at 37° C. 1 ml of theculture was induced with 1 mM IPTG. 2-4 hours later the 250 μl of eachculture was mixed with 250 μl acid washed glass beads and 250 μl Thornerbuffer with 5% βME and dye (8M urea, 100 mM Tris pH7.0, 10% glycerol, 2mM EDTA, 5% SDS). Samples were vortexed for one minute and heated to 65°C. for 10 minutes. 20 μl were loaded per lane on a 4%-12% PAGE gel(NOVEX). Gels were run in 1XMES buffer. The positive clones weredesignated pCZR225 and subjected to sequence analysis. Thepolynucleotide sequence of MBP-zalpha11 fusion is shown in SEQ ID NO:50.

B. Bacterial Expression of Human Huzalpha11/MBP-6H Fusion Polypeptide

One microliter of sequencing DNA was used to transform strain BL21. Thecells were electropulsed at 2.0 kV, 25 μF and 400 ohms. Followingelectroporation, 0.6 ml MM/CA with 100 mg/L Ampicillin.

Cells were grown in MM/CA with 100 μg/ml Ampicillin for two hours,shaking, at 37° C. 1 ml of the culture was induced with 1 mM IPTG. 2-4hours later the 250 μl of each culture was mixed with 250 μl acid washedglass beads and 250 μl Thorner buffer with 5% βME and dye (8M urea, 100mM Tris pH7.0, 10% glycerol, 2 mM EDTA, 5% SDS). Samples were vortexedfor one minute and heated to 65° C. for 10 minutes. 20 μl were loadedper lane on a 4%-12% PAGE gel (NOVEX). Gels were run in 1XMES buffer.The positive clones were used to grow up for protein purification of thehuzalpha11/MBP-6H fusion protein (Example 14, below).

Example 14 Purification of Huzalpha11/MBP-6H Soluble Receptor From E.coli Fermentation

Unless otherwise noted, all operations were carried out at 4° C. Thefollowing procedure was used for purifying huzalpha11/MBP-6H solublereceptor polypeptide. E. coli cells containing the pCZR225 construct andexpressing huzalpha11/MBP-6H soluble receptor (Example 13) were grown upin SuperBroth II (12 g/L Casien, 24 g/L Yeast Extract, 11.4 g/Ldi-potassium phosphate, 1.7 g/L Mono-potassium phosphate; BectonDickenson, Cockeysville, Md.), and frozen in 0.5% glycerol. Twenty gramsof the frozen cells in SuperBroth II+Glycerol were used to purify theprotein. The frozen cells were thawed and diluted 1:10 in a proteaseinhibitor solution (Extraction buffer) prior to lysing the cells andreleasing the huzalpha11/MBP-6H soluble receptor protein. The dilutedcells contained final concentrations of 20 mM Tris (JT Baker,Philipsburg, N.J.) 100 mM Sodium Chloride (NaCl, Mallinkrodt, Paris,Ky.), 0.5 mM pheynlmethylsulfonyl fluoride (PMSF, Sigma Chemical Co.,St. Louis, Mo.), 2 μg/ml Leupeptin (Fluka, Switzerland), and 2 μg/mlAprotinin (Sigma). A French Press cell breaking system (Constant SystemsLtd., Warwick, UK) with temperature of −7 to −10° C. and 30K PSI wasused to lyse the cells. The diluted cells were checked for breakage byA₆₀₀ readings before and after the French Press. The lysed cells werecentrifuged @ 18,000 G for 45 minutes to remove the broken cell debris,and the supernatant used to purify the protein. Total target proteinconcentrations of the supernatant was determined via BCA Protein Assay(Pierce, Rockford, Ill.), according to manufacturer's instructions.

A 25 ml column of Talon Metal Affinity resin (Clontech, Palo Alto,Calif.) (prepared as described below) was poured in a Bio-Rad, 2.5 cmD×10 cm H glass column. The column was packed and equilibrated bygravity with 10 column volumes (CVs) of Talon Equilibration buffer (20mM Tris, 100 mM NaCl, pH 8.0). The supernatant was batch loaded to Talonmetal affinity resin and was rocked overnight. The resin was poured backinto the column and was washed with 10 CV's of Talon Equilibrationbuffer by gravity, then gravity eluted with 140 ml of Elution buffer(Talon Equilibration buffer+200 mM Imidazole-Fluka Chemical). The taloncolumn was cleaned with 5 CVs of 20 mM 2-(N-Morhpholino) ethanesulfonicacid pH 5.0 (MES, Sigma), 5 CVs of distilled H₂O, then stored in 20%Ethanol/0.1% Sodium Azide. Fourteen ml fractions were collected over theentire elution chromatography and the fractions were read withabsorbance at 280 and 320 nM and BCA protein assay; the pass through andwash pools were also saved and analyzed. The protein elution fractionsof interest were pooled and loaded straight to Amylose resin (NewEngland Biolabs, Beverly, Mass.).

To obtain more pure huzalpha11/MBP-6H polypeptide, the talon affinityelution pooled fractions were subjected to Amylose resin (22 mls) at pH7.4. A 2.5 cm D×10 cm H Bio-Rad column was poured, packed andequilibrated in 10 CVs of Amylose equilibration buffer-20 mM Tris (JTBaker), 100 mM NaCl (Mallinkrodt), 1 mM PMSF (Sigma), 10 mMbeta-Mercaptoethanol (BME, ICN Biomedicals Inc., Aurora, Ohio) pH 7.4.The sample was loaded by gravity flow rate of 0.5 ml/min. The column waswashed for 10 CVs with Amylose equilibration buffer, then eluted with ˜2CV of Amylose equilibration buffer+10 mM Maltose (Fluka Biochemical,Switzerland) by gravity. 5 ml fractions were collected over the entirechromatography and absorbance at 280 and 320 nM were read. The Amylosecolumn was regenerated with 1 CV of distilled H₂O, 5 CVs of 0.1% (w/v)SDS (Sigma), 5 CVs of distilled H₂O, and then 5 CVs of Amyloseequilibration buffer.

Fractions of interest were pooled and dialyzed in a Slide-A-Lyzer(Pierce) with 4×4 L PBS pH 7.4 (Sigma) to remove low molecular weightcontaminants, buffer exchange and desalt. After the changes of PBS, thematerial harvested represented the purified huzalpha11/MBP-6Hpolypeptide. The purified huzalpha11/MBP-6H polypeptide was analyzed viaSDS-PAGE Coomassie staining and Western blot analysis with theanti-rabbit HRP conjugated antibody (Rockland, Gilbertsville, Pa.). Theconcentration of the huzalpha11/MBP-6H polypeptide was 1.92 mg/ml asdetermined by BCA analysis.

Purified huzalpha11/MBP-6H polypeptide was prepared for injection intorabbits and sent to R & R Research and Development (Stanwood, Wash.) forantibody production. Rabbits were injected to produce antianti-huzalpha11/MBP-6H serum (Example 15, below).

Example 15 Zalpha11 Polyclonal Antibodies

Polyclonal antibodies were prepared by immunizing two female New Zealandwhite rabbits with the purified huzalpha11/MBP-6H polypeptide (Example14), or the purified recombinant zalpha11CEE soluble receptor (Example11A). Corresponding polyclonal antibodies were designated rabbitanti-huzalpha11/MBP-6H and rabbit anti-huzalpha11-CEE-BHK respectively.The rabbits were each given an initial intraperitoneal (IP) injection of200 mg of purified protein in Complete Freund's Adjuvant (Pierce,Rockford, Ill.) followed by booster IP injections of 100 mg purifiedprotein in Incomplete Freund's Adjuvant every three weeks. Seven to tendays after the administration of the third booster injection, theanimals were bled and the serum was collected. The rabbits were thenboosted and bled every three weeks.

The zalpha11-specific polyclonal antibodies were affinity purified fromthe rabbit serum using an CNBr-SEPHAROSE 4B protein column (PharmaciaLKB) that was prepared using 10 mg of the purified huzalpha11/MBP-6Hpolypeptide (Example 14) per gram CNBr-SEPHAROSE, followed by 20×dialysis in PBS overnight. Zalpha11-specific antibodies werecharacterized by an ELISA titer check using 1 mg/ml of the appropriateprotein antigen as an antibody target. The lower limit of detection(LLD) of the rabbit anti-huzalpha11/MBP-6H affinity purified antibody isa dilution of 500 pg/ml. The LLD of the rabbit anti-huzalpha11-CEE-BHKaffinity purified antibody is a dilution of 50 pg/ml.

Example 16 Identification of Cells Expressing Zalpha11 Receptor UsingRT-PCR

Specific human cell types were isolated and screened for zalpha11expression by RT-PCR. B-cells were isolated from fresh human tonsils bymechanical disruption through 100 μm nylon cell strainers (Falcon™;Bectin Dickenson, Franklin Lakes, N.J.). The B-cell suspensions wereenriched for CD19+ B-cells by positive selection with VarioMACS VS+magnetic column and CD19 microbeads (Miltenyi Biotec, Auburn, Calif.) asper manufacturer's instructions. T-cells and monocytes were isolatedfrom human apheresed blood samples. CD3+ T-cells were purified by CD3microbead VarioMACS positive selection and monocytes were purified byVarioMACS negative selection columns (Miltenyi) as per manufacturer'sinstructions. Samples from each population were stained and analyzed byfluorescent antibody cell sorting (FACS) (Bectin Dickinson, San Jose,Calif.) analysis to determine the percent enrichment and resultingyields. CD19+ B-cells were approximately 96% purified CD3+ T-cells wereapproximately 95% purified, and monocytes were approximately 96%purified.

RNA was prepared, using a standard method in the art, from all threecell types that were either resting or activated. RNA was isolated fromresting cells directly from the column preparations above. The CD19+ andCD3+ cells were activated by culturing at 500,000 cells/ml in RPMI+10%FBS containing PMA 5 ng/ml (Calbiochem, La Jolla, Calif.) and Ionomycin0.5 ug/ml (Calbiochem) for 4 and 24 hours. The monocytes were activatedby culturing in RPMI+10% FBS containing LPS 10 ng/ml (Sigma St. LouisMo.) and rhIFN-g 10 ng/ml (R&D, Minneapolis, Minn.) for 24 hours. Cellswere harvested and washed in PBS. RNA was prepared from the cell pelletsusing RNeasy Midiprep™ Kit (Qiagen, Valencia, Calif.) as permanufacturer's instructions and first strand cDNA synthesis wasgenerated with Superscript II™ Kit (GIBCO BRL, Grand Island, N.Y.) asper manufacturers protocol.

Oligos ZC19907 (SEQ ID NO:20) and ZC19908 (SEQ ID NO:21) were used in aPCR reaction to screen the above described samples for a 1.2 kb fragmentcorresponding to zalpha11 message. PCR amplification was performed withTaq Polymerase (BRL Grand Island N.Y.), and conditions as follows: 35cycles of 95° C. for 1 min, 60° C. for 1 min., 72° C. for 30 sec.; 1cycle at 72° C. for 10 min.; and 4° C. soak. 10 ul of each 50 μlreaction volume was run on a 2% agarose 1XTAE gel to identify resultantproducts. PCR products were scored as (−) for no product, (+) for bandvisible, (++) increased presence of band and (+++) being the mostpredominant band, with results shown in Table 5 below.

TABLE 5 cDNA Source Activation PCR Product CD19+ cells  0 hr resting + 4 hr activated ++ 24 hr activated +++ CD3+ cells  0 hr resting −  4 hractivated ++ 24 hr activated − monocytes  0 hr resting − 24 hr activated−

These results indicated that zalpha11 message is present in restinghuman CD19+ B-cells and increases with mitogenic activation. It alsoappears to be expressed by human CD3+ T-cells only after 4 houractivation. There was no apparent message in either resting or activatedhuman monocytes.

Example 17 Zalpha 11 Immunohistochemistry

A. Cell and Tissue Preparations

Positive control tissues consisted of BaF3 cells transfected withzalpha11 (Example 7) and lymphoid tissues known to express zalpha11including mouse lymph node, spleen and thymus received from HSD (HarlanSprague Dawley, Indianapolis, Ind.), monkey lymph node and spleenreceived from Regional Primate Research Center (University ofWashington, Seattle, Wash.), human lymph node and spleen received fromCHTN (Cleveland, Ohio). Negative controls performed on each tissuesample included: (1) untransfected BaF3 cells, (2) liver and brain frommouse and human known not to express zalpha11, (3) staining withantibody dilution buffer (Ventann Bioteck Systems, Tucson Ariz.) in theabsence of primary antibody, and (4) using zalpha11 soluble protein incompetition experiments.

Other cell samples were examined. Both non-stimulated and stimulatedHL60 cells were assayed. HL60 cells are a promyelocytic cell line, whichcan be differentiated into myeloid or granulocyte lineages withdifferent reagents. Stimulated HL60 samples were prepared as follows:(1) HL60 cells were treated with 10 ng/ml of phorbol-myristate-acetate(PMA) (Sigma, St. Louis, Mo.) for 48 hours to differentiate intomonocyte lineage cells; and (2) HL60 cells treated with 1.25% DMSO(Sigma) for 4 days to differentiate into neutrophil-like cells. Inaddition, human polymorphonuclear (PMN) cells, human granulocytes, humanperipheral blood lymphocytes (PBL) and human monocytes from fresh humanblood were examined (prepared in house using routine methods in theart). The cells and tissues described above were fixed overnight in 10%NBF (Surgipath, Richmond, Ill.), and embedded in parapalst X-tra (OxfordScientific, St. Louis, Mo.), and sectioned at 5 μm with a Reichart-Jung2050 microme (Leica Instruments GmbH, Nussloch, Germany).

B Immunohistochemistry

Tissue slides were deparaffinized, hydrated to buffer (water), andsubjected to steam HIER treatment in Antigen Retrieval Citra buffer(BioGenex, San Roman, Calif.) for 20 minutes. 5% normal goat serum(Vector, Burlingame, Calif.) was used to block non-specific binding for10 minutes Immunocytochemical screening analyses were performed usingpolyclonal antibodies to zalpha11 soluble receptor protein (rabbitanti-huzalpha11-MBP-6H and rabbit anti-huzalpha11-CEE-BHK; see, Example15) as the primary antibodies, at dilutions of 1:200 and 1:400respectively. Biotin conjugated goat anti-rabbit IgG (Vector; Cat. No.BA-1000, 1.5 mg/ml) was used as the secondary antibody at dilution of1:200. In separate samples, protein competition was performed by usingadditional zalpha11CEE soluble receptor protein (in 10× fold excess)(Example 11A) to the primary antibody to pre-block primary antibodyimmunoreaction. This competition was used as a control for the rabbitpolyclonal antibody specificity to zalpha11. Detection was performed onthe Ventana ChemMate 500 instrument using a ChemMate DAB Kit (labeledStreptavidin-Biotin Kit with application of a streptavidin-horseradishperoxidase conjugate, and DAB substrate) according to manufacturer'sinstruction and using the manufacturer's hematoxylin counterstain for 30seconds (Ventana Biotek Systems, Tucson, Ariz.).

High expression of zalpha11 was observed in the PMA-activated HL60cells. Low level expression was observed in PBL and HL60 cells withoutstimulation. A subset of cells in the spleen, thymus and lymph node ofmouse showed positive staining Lymph node and spleen of both human andmonkey, and HL60 cells with DMSO stimulation showed minimal or nostaining. The signal seen in the cells and tissues was mostly competedout by using the excess zalpha11 soluble receptor protein. The negativecontrol tissues of brain and liver showed no staining.

Example 18 Identifying Peripheral Blood Mononuclear Cells (PBMNC's) thatExpress Zalpha11 Receptor Using Polyclonal Rabbit Anti-sera to Zalpha11Soluble Receptor

200 ml fresh heparinized blood was obtained from a normal donor. Bloodwas diluted 1:1 in PBS, and separated using a Ficoll-Paque PLUS gradient(Pharmacia Biotech, Uppsala, Sweden), and the lymphocyte interfacecollected. Cells were washed 2× in PBS and resuspended in RPMI+5% FBSmedia at a concentration of 2×10⁶ cells/ml.

In order to determine whether expression of zalpha11 receptor isaffected by the activation state of the lymphocyte cells, i.e., betweenresting and activated cells several stimulation conditions were used: 1)un-stimulated, i.e., media alone (RPMI+5% FBS media); 2) stimulated withPMA 10 ng/ml+Ionomycin 0.5 μg/ml (both from Calbiochem); and 3) PHAactivation (phytohemagglutinin-P, Difco/VWR). The cells were incubatedat 37° C. for 17 hours then collected for staining to detect expressionof zalpha11 receptor.

An indirect staining protocol was used. Briefly, the human lymphocytecells were suspended in staining buffer (PBS+0.02% NaN3+BSA 1% normalhuman serum 2%) and plated at 2×10⁵ cells in 50 μl/well in a 96 wellplate. Antibodies to the zalpha11CEE soluble receptor (Example 15) wereused to determine whether they co-stained with a B-cell (CD-19), T-cell(CD-3) or monocyte marker (CD-14) on the isolated human lymphocytes. Arabbit polyclonal sera to zalpha11 soluble receptor (Rbanti-huzalpha11-CEE-BHK) (Example 15) at 10 μg/ml was used as theantibody to identify zalpha11 on the lymphocytes. A secondary antibody,goat anti-rabbit Ig-FITC (Biosource, Camarillo, Calif.), was used tovisualize the Rb anti-huzalpha11-CEE-BHK antibody binding to thezalpha11 receptors. Other antibodies were simultaneously used to stain Tcells (CD3-PE; PharMingen, San Diego, Calif.), B cells (CD19-PE)(PharMingen), and monocytes (CD-14-PE) (PharMingen) in order to identifyco-staining of the anti-zalpha11 receptor antibody on these cell types.Various controls were used to determine non-specific binding andbackground levels of staining: (1) an irrelevant rabbit polyclonal serawas used as a non-specific control; and (2) secondary antibody alone wasused to determine background binding of that reagent. Purified,zalpha11CEE soluble receptor (Example 11) was used in about a 10-foldexcess as a competitive inhibitor to verify the specificity of therabbit anti-huzalpha11-CEE-BHK antibody to zalpha11 soluble receptor.

After plating the cells and adding the primary and co-stainingantibodies, the cells were incubated on ice for 30 minutes, washed 2×with staining buffer, and stained with the secondary antibody, goatanti-rabbit Ig-FITC (Biosource), for 30 minutes on ice. Cells werewashed 2× staining buffer, and resuspended at 200 μl per well instaining buffer containing the viability stain 7AAD at about 1 μg/mlfinal concentration (Sigma, St. Louis, Mo.). Samples were read on theFACS-Caliber (Becton-Dickinson, San Jose, Calif.) and viable cellsanalyzed.

The rabbit polyclonal to zalpha11 receptor stained resting B cells. Thesignal on resting B cells was brighter than the signal achieved usingthe irrelevant rabbit sera, and the signal was diminished to a greaterextent on B cells than on T cells with the addition of excesszalpha11-CEE soluble receptor. This experiment was repeated usingseparated B and T cells, and the results were very similar. Again thestaining with the polyclonal rabbit anti-huzalpha11-CEE-BHK antibody tozalpha11 receptor was highest on resting B cells.

Example 19 Zalpha11 Receptor Expression in Various Tissues UsingReal-Time Quantitative RT/PCR

A. Primers and Probes for Quantitative RT-PCR-

Real-time quantitative RT-PCR using the ABI PRISM 7700 SequenceDetection System (PE Applied Biosystems, Inc., Foster City, Calif.) hasbeen previously described (See, Heid, C. A. et al., Genome Research6:986-994, 1996; Gibson, U. E. M. et al., Genome Research 6:995-1001,1996; Sundaresan, S. et al., Endocrinology 139:4756-4764, 1998. Thismethod incorporates use of a gene specific probe containing bothreporter and quencher fluorescent dyes. When the probe is intact thereporter dye emission is negated due to the close proximity of thequencher dye. During PCR extension using additional gene-specificforward and reverse primers, the probe is cleaved by 5′ nucleaseactivity of Taq polymerase which releases the reporter dye from theprobe resulting in an increase in fluorescent emission.

The primers and probes used for real-time quantitative RT-PCR analysesof zalpha11 expression were designed using the primer design softwarePRIMER EXPRESS™ (PE Applied Biosystems, Foster City, Calif.). Primersfor human zalpha11 were designed spanning an intron-exon junction toeliminate amplification of genomic DNA. The forward primer, ZC22,277(SEQ ID NO:59) and the reverse primer, ZC22,276 (SEQ ID NO:60) were usedin a PCR reaction (below) at about 300 nM concentration to synthesize a143 bp product. The corresponding zalpha11 TAQMAN® probe, designatedZG31 (SEQ ID NO:61) was synthesized and labeled by PE AppliedBiosystems. The ZG31 probe was labeled at the 5′ end with a reporterfluorescent dye (6-carboxy-fluorescein) (FAM) (PE Applied Biosystems)and at the 3′ end with a quencher fluorescent dye(6-carboxy-tetramethyl-rhodamine) (TAMRA™) (PE Applied Biosystems).

As a control to test the integrity and quality of RNA samples tested,all RNA samples (below) were screened for rRNA using a primer and probeset ordered from PE Applied Biosystems (cat No. 4304483). The kitcontains an rRNA forward primer (SEQ ID NO:66) and the rRNA reverseprimer (SEQ ID NO:67), rRNA TAQMAN® probe (SEQ ID NO:68) The rRNA probewas labeled at the 5′ end with a reporter fluorescent dye VIC (PEApplied Biosystems) and at the 3′ end with the quencher fluorescent dyeTAMRA™ (PE Applied Biosystems). The rRNA results also serve as anendogenous control and allow for the normalization of the zalpha11 mRNAexpression results seen in the test samples.

RNA samples from human CD3, CD19 and monocyte cell types were preparedand described as per Example 16 above. Control RNA was prepared, usingRNeasy Miniprep™ Kit (Qiagen, Valencia, Calif.) as per manufacturer'sinstructions, from approximately 10 million BaF3 cells expressing humanzalpha11 receptor (Example 7).

B. Real-time Quantitative RT-PCR-

Relative levels of zalpha11 mRNA were determined by analyzing total RNAsamples using the one-step RT-PCR method (PE Applied Biosystems). TotalRNA from BaF3 cells expressing human zalpha11 receptor was isolated bystandard methods and used to generate a standard curve used forquantitation. The curve consisted of 10-fold serial dilutions rangingfrom 2.5−2.5×10⁻⁴ ng/ml for the rRNA screen and 250-0.025 ng/ml for thezalpha11 screen with each standard curve point analyzed in triplicate.The total RNA samples from the human CD3, CD19 and monocyte cells werealso analyzed in triplicate for human zalpha11 transcript levels and forlevels of rRNA as an endogenous control. In a total volume of 25 μl,each RNA sample was subjected to a One-Step RT-PCR reaction containing:approximately 25 ng of total RNA in buffer A (50 mM KCL, 10 mMTris-HCL); the internal standard dye, carboxy-x-rhodamine (ROX));appropriate primers (approximately 50 nM rRNA primers (SEQ ID NO:66 andSEQ ID NO:67) for the rRNA samples; and approximately 300 nM ZC22,277(SEQ ID NO:59) and ZC22,276 (SEQ ID NO:60) primers for zalpha11samples); the appropriate probe (approximately 50 nM rRNA TAQMAN® probe(SEQ ID NO:68) for rRNA samples, approximately 100 nM ZG31 (SEQ IDNO:61) probe for zalpha11 samples); 5.5 mM MgCl₂; 300 μM each d-CTP,d-ATP, and d-GTP and 600 μM of d-UTP; MuLV reverse transcriptase (0.25U/μl); AMPLITAQ GOLD™ DNA polymerase (0.025 U/μl) (PE AppliedBiosystems); and RNase Inhibitor (0.4 U/μl) (PE Applied Biosystems). PCRthermal cycling conditions were as follows: an initial reversetranscription (RT) step of one cycle at 48° C. for 30 minutes; followedby an AMPLITAQ GOLD™ DNA polymerase (PE Applied Biosystems) activationstep of one cycle at 95° C. for 10 minutes; followed by 40 cycles ofamplification at 95° C. for 15 seconds and 60° C. for 1 minute.

Relative zalpha11 RNA levels were determined by using the Standard CurveMethod as described by the manufacturer, PE Biosystems (User BulletinNo. 2: ABI Prism 7700 Sequence Detection System, Relative Quantitationof Gene Expression, Dec. 11, 1997). The rRNA measurements were used tonormalize the zalpha11 levels and the resting CD3+ RNA sample was usedas a calibrator. Resting CD3 was arbitrarily chosen as the calibratorand given a value of 1.00. The rest of the samples were comparedrelative to the calibrator. Data are shown in Table 6 below.

TABLE 6 4 hr Sample Resting Stimulation 24 hr Stimulation CD3 1.00 15.2716.70 CD19 20.14 65.08 25.42 Monocytes 0.05 no data 0.26

There was a 15-fold increase in zalpha11 receptor expression in CD3+ at4 and 24 hrs. Resting CD19 had 20 fold increase in receptor expressionrelative to resting CD3+. There was a 3 fold increase with 4 hrstimulation that fell back to resting levels by 24 hrs. Monocytes showedno detectable zalpha11 receptor expression in this assay.

Example 20 Identification of Cells Expressing Zalpha11 Receptor Using insitu Hybridization

Specific human tissues were isolated and screened for zalpha11expression by in situ hybridization. Various human tissues prepared,sectioned and subjected to in situ hybridization included thymus,spleen, tonsil, lymph node and lung. The tissues were fixed in 10%buffered formalin and blocked in paraffin using standard techniques(Example 17). Tissues were sectioned at 4 to 8 microns. Tissues wereprepared using a standard protocol (“Development of non-isotopic in situhybridization” at http://dir.niehs.nih.gov/dirlepish.html). Briefly,tissue sections were deparaffinized with HistoClear (NationalDiagnostics, Atlanta, Ga.) and then dehydrated with ethanol. Next theywere digested with Proteinase K (50 μg/ml) (Boehringer Diagnostics,Indianapolis, Ind.) at 37° C. for 2 to 20 minutes. This step wasfollowed by acetylation and re-hydration of the tissues.

Two in situ probes generated by PCR were designed against the humanzalpha11 sequence. Two sets of oligos were designed to generate probesfor separate regions of the zalpha11 cDNA: (1) Oligos ZC23,684 (SEQ IDNO:62) and ZC23,656 (SEQ ID NO:63) were used to generate a 413 bp probefor zalpha11; and (2) Oligos ZC23,685 (SEQ ID NO:64) and ZC23,657 (SEQID NO:65) were used to generate a 430 bp probe for zalpha11. The secondprobe is 1500 bp 3′ of the first zalpha11 probe. The antisense oligofrom each set also contained the working sequence for the T7 RNApolymerase promoter to allow for easy transcription of antisense RNAprobes from these PCR products. The PCR reaction conditions were asfollows: 30 cycles at 94° C. for 30 sec, 60° C. for 1 min., 72° C. for1.5 min. The PCR products were purified by Qiagen spin columns followedby phenol/chloroform extraction and ethanol precipitation. Probes weresubsequently labeled with digoxigenin (Boehringer) or biotin(Boehringer) using an In Vitro transcription System (Promega, Madison,Wis.) as per manufacturer's instruction.

In situ hybridization was performed with a digoxigenin- orbiotin-labeled zalpha11 probe (above). The probe was added to the slidesat a concentration of 1 to 5 μmol/ml for 12 to 16 hours at 55-60° C.Slides were subsequently washed in 2×SSC and 0.1×SSC at 50° C. Thesignals were amplified using tyramide signal amplification (TSA) (TSA,in situ indirect kit; NEN) and visualized with Vector Red substrate kit(Vector Lab) as per manufacturer's instructions. The slides were thencounter-stained with hematoxylin (Vector Laboratories, Burlingame,Calif.).

A signal was seen in the thymus, tonsil, lung, and lymph node. Thepositive-staining cells appeared to be lymphocytes and related cells.

Example 21 Isolation of the Mouse Zalpha11 Receptor

A. Mouse Genomic Library Screen

An initial partial mouse zalpha11 sequence was obtained by probing amouse genomic library with a human zalpha11 receptor polynucleotideprobe containing the entire cDNA. The human zalpha11 cDNA was generatedby PCR with ZC19,905 (SEQ ID NO:36) and ZC19,906 (SEQ ID NO:37) primersand a plasmid containing full length human zalpha11 (e.g., Example 1)was used for the template. The PCR reaction conditions were as follows:35 cycles at 98° C. for 1 min., 68° C. for 1 min., and 72° C. for 2min.; followed by one cycle at 72° C. for 10 min. The PCR product wasrun on a 1% low melting point agarose (Boerhinger Mannheim) and theapproximately 1.5 kb human zalpha11 cDNA isolated using QIAQUICK™ gelextraction kit (Qiagen) as per manufacturer's instructions. This humanzalpha11 cDNA was used to screen a mouse genomic DNA library (below).

The mouse genomic DNA library used was emb13 SP6/T7 lambda BamHI clonedlibrary (Clontech, Palo Alto, Calif.). This library representing 7.2×10⁵pfu was plated onto an E. coli K802 host lawn on 24 NZY plates. Plaquelifts were performed using HYBOND™-N filters (Amersham Pharmacia,Buckinghamshire, England, UK) as per manufacturer's instructions. Thefilters were denatured in 1.5 M NaCl and 0.5 M NaOH for 10 min. and thenneutralized in 1.5 M NaCl and 0.5 M Tris-HCL (pH 7.2) for 10 min. TheDNA was affixed to the filter using a STRATALINKER® UV crosslinker(Stratagene) at 1200 joules. The filters were pre-washed to remove celldebris at 65° C. in pre-wash buffer (0.25×SSC, 0.25% SDS and 1 mM EDTA),changing solution three times for a total of 45 min. The filters wereprehybridized overnight at 50° C. in EXPRESSHYB™ solution (Clontech)containing 0.1 mg/ml denatured salmon sperm DNA. Approximately 50 ng ofthe purified human zalpha11 cDNA (above) was labeled with ³²P using theREDIPRIME™ II Random Prime Labeling System (Amersham Pharmacia) as permanufacturers instructions. Unincorporated radioactivity was removedfrom the zalpha11 cDNA probe using a NUCTRAP™ push column (Stratagene,La Jolla, Calif.). Filters were hybridized in EXPRESSHYB™ solution(Clontech) containing about 0.5 to about 1×10⁶ cpm/ml zalpha11 cDNAprobe, about 0.1 mg/ml denatured salmon sperm DNA and denatured 0.5μg/ml cot-1 DNA. Hybridization took place overnight at 50° C. Filterswere washed in 2×SSC, 0.1% SDS at room temperature for 2 hours (changingwash several times) then the temperature was raised to 60° C. for onehour (changing buffer once). Overnight exposure at −80° C. showed 6plaques representing primary isolates.

To obtain secondary plaque isolates, the 6 plaques representing primaryisolates were picked with a Pasteur pipette and eluted overnight at 4°C. in 1 ml SM (0.1 M NaCl, 50 mM Tris pH 7.5, 10 mM MgSO₄, 0.02%gelatin) containing a few drops of chloroform. After determining phagetiters, about 12.5× the estimated amount of phage in the original plug(12.5× coverage) of 6 primary isolates was plated on a lawn of E. coliK802 cells embedded in 10 mM MgSO₄/NZY top agarose on NZY maxi plates,and grown overnight at 37° C. Plaque lifts were done using HYBOND™-Nfilters (Amersham Pharmacia) as per manufacturer's instructions. Filterswere fixed as per above. The second round filters were pre-washed toremove cell debris at 65° C. in pre-wash buffer (2×SSC, 0.1% SDS and 1mM EDTA), changing solution three times for a total of 45 min. Thesecond round filter lifts were then prehybridized, and the zalpha11 cDNAprobe prepared as described above.

The second round filters were hybridized as above in EXPRESSHYB™solution (Clontech) containing about 10⁶ cpm/ml zalpha11 cDNA probecontaining about 0.1 mg/ml denatured salmon sperm DNA. Hybridizationtook place overnight at 50° C. Wash conditions described above for theprimary screen were repeated for this secondary screen. After anovernight exposure at −80° C., two of the 6 original primary plaquesisolates were verified as positive in the secondary screen. Positiveplaques hybridizing to human zalpha11 cDNA in the secondary screen werepicked with a Pasteur pipette and designated 7b1 and 20b1.

The isolated plaques No. 7b1 and 20b1 were eluted in 200 μl SM overnightat 4° C. Serial 10-fold serial dilutions ranging from 10⁻² to 10⁻⁶ ofeach isolate were plated on host E. coli K802 cells to determine thetiter. Isolate 20b1 had a titer of 4×10³ pfu/μl and was further pursued.4 plates were prepared by plating 10⁵ pfu/plate on confluent host E.coli K802 cells in order to make a phage DNA prep. Plates were grown at37° C. for about 6.5 hours until phage lysis was starting to getconfluent. The phage was then eluted overnight at 4° C. in 12 ml of SMper plate. Plates were then shaken at room temperature one hour, thesupernatant was removed; 1% chloroform was added and supernatant wasshaken again for 15 min. The 20b1 phage DNA was prepped using theWIZARD™ Lambda Preps DNA Purification System (Promega, Madison, Wis.;sections IV and VI).

Samples of 20b1 phage DNA were cut with several restriction enzymes togenerate DNA fragments for Southern blotting. The digests were run on a1% TBE agarose gel. The gel was soaked in 0.25 M HCl for 30 min.; rinsedin distilled H20; soaked in 0.5M NaOH and 1.5 M NaCl for 40 min. withone solution change and neutralized in 1.5 NaCl and 0.5 Tris-HCL (pH7.2) for 40 min. with one solution change. A TURBOBLOTTER™ RapidDownward Transfer System (Schleicher & Schuell, Keene, N.H.) was set upto transfer the DNA onto a NYTRAN®/BA-S membrane (Schleicher & Schuell)overnight. The DNA was affixed to the NYTRAN® membrane using aSTRATALINKER® UV crosslinker (Stratagene, La Jolla, Calif.) at 1200joules. The blot was prehybridized as described above. About 50 ng ofthe human zalpha11 cDNA was labeled and purified for a probe, asdescribed above. Filters were hybridized as above in EXPRESSHYB™solution (Clontech) containing about 10⁶ cpm/ml zalpha11 cDNA probe andabout 0.1 mg/ml denatured salmon sperm DNA. Hybridization took placeovernight at 50° C. The blot was washed as described above and exposedto film overnight at −80° C.

The Southern showed a DNA fragment generated from a BamHI/StuI digestwhich hybridized to the human zalpha11 cDNA probe in the expected sizerange of 1.3 to 1.6 kb. This fragment was pursued. Approximately 3 μg of20b1 lambda DNA was cut with 20 units of BamHI (Boehringer Mannheim,Indianapolis, Ind.) and 20 Units StuI (NEB, Beverly, Mass.) for 2 hoursat 37° C. The digest was run on a 1% TBE gel and a 1.3 kb doublet and1.6 kb doublet bands were excised from the gel and the DNA was extractedfrom the agarose using the QIAQUICK™ Gel Extraction Kit (Qiagen,Valencia, Calif.). Due to the low yield of DNA from the prep, it was notpossible to determine by additional restriction digest analysis whetherfragments which hybridized to the human zalpha11 cDNA probe wereBamHI/StuI or StuI/StuI fragments. Thus, blunt ligations using 5 μl ofthe 1.3 kb doublet fragment and 5 μl of the 1.6 kb doublet fragment weredone using the ZERO BLUNT® PCR Cloning Kit (Invitrogen, Carlsbad,Calif.). The blunt ligation yielded positive clones with both of the 1.6kb fragments and one of the 1.3 kb fragments. These clones were digestedwith EcoRI (Life Technologies) which flanks the T-overhang site wherethe 1.6 and 1.3 kb fragments were inserted. Another Southern blot wasperformed to determine which was the original fragment hybridizing tothe human zalpha11 cDNA probe. The 1% TBE gel was treated and the DNAwas transferred to the NYTRAN® blot as described above.

The blot was prehybridized as above in 10 ml of hybridization solution.A different human zalpha11 polynucleotide probe was prepared. Anotherfull length zalpha11 cDNA human zalpha11 fragment was generated for useas a probe by PCR with the oligos ZC19,905 (SEQ ID NO: 36) and ZC20,097(SEQ ID NO: 27). The PCR reaction conditions were as follows: 95° C. for1 min.; 35 cycles of 95° C. for 1 min., 55° C. for 1 min., and 72° C.for 2 min.; followed by one cycle at 72° C. for 10 min. The PCR productwas run on a 1% low melting point agarose (Boerhinger Mannheim) and theapproximately 1.5 kb human zalpha11 cDNA isolated using QIAQUICK™ gelextraction kit (Qiagen) as per manufacturer's instructions. About 50 ngof this isolated human zalpha11 cDNA fragment was labeled with ³²P andpurified as described above. Filters were hybridized as above inEXPRESSHYB™ solution (Clontech) containing about 10⁶ cpm/ml zalpha11cDNA probe, about 0.1 mg/ml denatured salmon sperm, and denatured 0.5μg/ml cot-1 DNA. Hybridization and washing was as described above. Theblot was exposed to film 1.5 hours at −80° C. and the 1.3 kb insert wasstrongly hybridizing to the human zalpha11 probe.

This clone was sequenced and found to contain a mouse zalpha11 3′ codingexon with a termination codon and upstream intron sequence. Sequencingprimers used were: ZC3,424 (SEQ ID NO:86), ZC694 (SEQ ID NO:87),ZC24,399 (SEQ ID NO:88), and ZC24,400 (SEQ ID NO:89). The genomicsequence of mouse zalpha11 including the 3′ exon is shown in SEQ IDNO:69. The 3′ exon coding sequence starts at nucleotide 543 and ends atnucleotide 1262 in SEQ ID NO:69, encoding a 240 amino acid exon (SEQ IDNO:70).

B. PCR Screen of Mouse cDNA Panel

A panel of available in-house and commercial mouse cDNAs (Clontech; Lifetechnologies, Gaithersburg, Md.) was screened by PCR using ZC24,432 (SEQID NO:71) and ZC24,433 (SEQ ID NO:72) as primers (about 20 pmol each).The PCR reaction conditions were as follows: 94° C., 2 min.; 32 cyclesof 94° C. for 20 sec., 64° C. for 30 sec., and 72° C. for 30 sec.;followed by one cycle at 72° C. for 5 min. Mouse spleen, dendriticcells, neonatal skin, bone marrow, wild type BaF3 cells, EL4 cells, andlung showed strong PCR products of the predicted 450 bp size.

C. 5′ Nested RACE

5′ RACE reactions were performed using 20 μmol each of primers ZC9,739(SEQ ID NO:73) and ZC24,434 (SEQ ID NO:74) and CD90+ selected mousespleen marathon cDNA as a template. The marathon cDNA was prepared usinga Marathon cDNA Amplification Kit (Clontech) according to themanufacturer's instructions. The PCR reaction conditions were asfollows: 94° C. for 1 min.; 5 cycles of 94° C. for 20 sec., and 70° C.for 1.5 min.; followed by 25 cycles of 94° C. for 20 sec., 64° C. for 20sec., and 70° C. for 1.5 min.; followed by one cycle at 72° C. for 5min.

To enrich for mouse zalpha11 5′ RACE product, a nested 5′ RACE reactionwas performed using PCR reaction conditions as described above for theinitial 5′RACE, except using nested primers ZC24,431 (SEQ ID NO:75) andZC9,719 (SEQ ID NO:76), and one μl of a 1/20 dilution of the initial 5′RACE reaction (above) as a template. The products were purified by gelelectrophoresis, the DNA was eluted using the Qiaex II Agarose GelExtraction Kit (Qiagen) and subcloned using the TOPO TA Cloning Kit(Invitrogen). Positive clones were identified by colony PCR using 10pmol each of ZC24,431 (SEQ ID NO:75) and ZC24,511 (SEQ ID NO:77). ThePCR reaction conditions were as follows: 94° C., 2 min.; 35 cycles of94° C. for 20 sec., 64° C. for 20 sec., and 72° C. for 30 sec.; followedby one cycle at 72° C. for 5 min. Two subclones from each of the nested5′RACE reactions were sequenced. All the clones contained some zalpha11sequence but none were complete. A compiled sequence was generated fromthe incomplete 5′RACE clones and the 3′ exon sequence (SEQ ID NO:70)representing a preliminary partial sequence of the mouse zalpha11polynucleotide and corresponding polypeptide. The preliminary sequenceof the partial mouse zalpha11 cDNA is show in SEQ ID NO:78 (5′ end) andSEQ ID NO:80 (3′ end); there was approximately 330 nucleotides of yetunknown sequence between SEQ ID NO:78 (5′ end) and SEQ ID NO:80 (3′ end)to comprise the entire mouse zalpha11 cDNA (see below). Thecorresponding amino acid sequences for SEQ ID NO:78 and SEQ ID NO:80 areshown in SEQ ID NO:79 (N-terminus) and SEQ ID NO:81 (C-terminus)respectively.

D. Full Length PCR

Primers were designed from the mouse upstream UTR of the initiation Metand downstream of the termination codon for full length PCR. 20 pmoleach of primers ZC24,616 (SEQ ID NO:82) and ZC24,615 (SEQ ID NO:83) wereused in PCR reactions using a mouse dendritic cell marathon cDNA or aneonatal mouse skin in-house cDNA library as a template. PCR reactionconditions were as follows: 94° C., 1 min.; 30 cycles of 94° C. for 20sec., and 66° C. for 2 min.; followed by one cycle at 72° C. for 5 min.The PCR products were purified by gel electrophoresis, and the cDNA waseluted using the QIAQUICK™ Gel Extraction Kit and subcloned using the TACLONING® Kit (Invitrogen). 2 subclones from each PCR reaction weresequenced. Sequencing primers used were: ZC694 (SEQ ID NO:87), ZC3,424(SEQ ID NO:86), ZC24,431 (SEQ ID NO:75), ZC24,511 (SEQ ID NO:77),ZC24,806 (SEQ ID NO:90), and ZC24,807 (SEQ ID NO:91). The sequence ofthe full length mouse zalpha11 cDNA is show in SEQ ID NO:84. Thecorresponding amino acid sequence is shown in SEQ ID NO:85.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A method of detecting, in a test sample, the presence of a modulatorof zalpha11 polypeptide activity, the method comprising: (a) culturing acell into which has been introduced an expression vector comprising thefollowing operably linked elements: (i) a transcription promoter; (ii) aDNA segment encoding a polypeptide consisting of an amino acid sequenceas shown in SEQ ID NO:2 from amino acid number 20 (Cys), to amino acidnumber 538 (Ser); and (iii) a transcription terminator, wherein thepromoter is operably linked to the DNA segment, and the DNA segment isoperably linked to the transcription terminator; and wherein the cellexpresses the polypeptide encoded by the DNA segment in the presence andabsence of a test sample; (b) comparing levels of activity of thepolypeptide in the presence and absence of a test sample, by abiological or biochemical assay, wherein the biological or biochemicalassay comprises the zalpha11 polypeptide exhibiting cell proliferationactivity or signal transduction activity when exposed to activated CD3+selected human T-cell conditioned media, thereby detecting the presenceof the modulator of zalpha11 polypeptide activity; and (c) determiningfrom the comparison the presence of the modulator of zalpha11polypeptide activity in the test sample.
 2. The method of claim 1,wherein the biological or biochemical assay comprises an assay whereinthe zalpha11 polypeptide is membrane bound within a cultured cell, andthe detecting step comprises measuring a biological response in thecultured cell.
 3. The method according to claim 2, wherein thebiological response is cell proliferation, signal transduction activity,or activation of transcription of a reporter gene.
 4. The methodaccording to claim 1, wherein the test sample is a blood sample,cell-conditioned culture media, or a tissue extract.
 5. The methodaccording to claim 4, wherein the tissue extract is from a tumor orleukemia.
 6. A method of detecting, in a test sample, the presence of amodulator of zalpha11 polypeptide activity, the method comprising: (a)culturing a cell into which has been introduced an expression vectorcomprising the following operably linked elements: (i) a transcriptionpromoter; (ii) a DNA segment encoding a polypeptide consisting of anamino acid sequence as shown in SEQ ID NO:2 from amino acid number 20(Cys), to amino acid number 237 (His), and wherein the polypeptidefurther comprises a transmembrane domain and an intracellular domainfrom a heterologous cytokine receptor; and (iii) a transcriptionterminator, wherein the promoter is operably linked to the DNA segment,and the DNA segment is operably linked to the transcription terminator;and wherein the cell expresses the polypeptide encoded by the DNAsegment in the presence and absence of a test sample; (b) comparinglevels of activity of the polypeptide in the presence and absence of atest sample, by a biological or biochemical assay, wherein thebiological or biochemical assay comprises the zalpha11 polypeptideexhibiting cell proliferation activity or signal transduction activitywhen exposed to activated CD3+ selected human T-cell conditioned media;and thereby detecting the presence of the modulator of zalpha11polypeptide activity; and (c) determining from the comparison, thepresence of the modulator of zalpha11 polypeptide activity in the testsample.
 7. The method of claim 6, wherein the biological or biochemicalassay comprises an assay wherein the zalpha11 polypeptide is membranebound within a cultured cell, and the detecting step comprises measuringa biological response in the cultured cell.
 8. The method according toclaim 7, wherein the biological response is cell proliferation, signaltransduction activity, or activation of transcription of a reportergene.
 9. The method according to claim 6, wherein the test sample is ablood sample, cell-conditioned culture media, or a tissue extract. 10.The method according to claim 9, wherein the tissue extract is from atumor or leukemia.
 11. The method according to claim 6, wherein theheterologous cytokine receptor is a class I cytokine receptor.